Name: a western blotting protocol for small numbers of hematopoietic stem cells
Uploaded: 2018-08-22T12:30:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

<p class="jove_content">National Institutes of Health grant R01 CA149976 (N.A.S) supported this work.</p>

<p class="jove_content">All procedures must be performed in accordance with institutional animal use and care guidelines. The procedure was developed for the analysis of murine hematopoietic stem and progenitor cells (HSCs and HPs), but can be adapted for the analysis of other cell populations.</p>
<p class="jove_title">1. Flow Cytometry Isolation of Murine HSCs and HPs</p>
<ol>
	<li>Harvest murine bone marrow cells as described in the literature<sup class="xref">6</sup>.<br />
	NOTE: In data presented in <strong class="xfig">Figure 1</strong> and <strong class="xfig">Figure 2</strong>, bone marrow was harvested from C57BL/6J mice.</li>
	<li>Perform lineage depletion of bone marrow cells using a mouse lineage depletion kit, following the manufacturer&#39;s instructions.</li>
	<li>Incubate the cells with antibodies against the cell surface markers of interest as described<sup class="xref">7</sup>. In the experiments shown in <strong class="xfig">Figure 1</strong> and <strong class="xfig">Figure 2</strong>, antibodies to Sca-1, Kit, Flt3, and lineage (Lin) markers Mac1, Gr1, CD3e, B220, and Ter119 are used.</li>
	<li>Sort 2,000 - 20,000 Lin<sup>- </sup>Sca1<sup>+</sup>Kit<sup>+</sup> Flt3<sup>-</sup> cells (HSCs) or Lin<sup>-</sup>Sca1<sup>-</sup>Kit<sup>+</sup> cells (HPs) into 1.5 mL Eppendorf tubes containing 0.1 mL of phosphate buffered saline (PBS) with 2% fetal bovine serum (FBS).<br />
	NOTE: For the data shown in <strong class="xfig">Figure 1</strong> and <strong class="xfig">Figure 2</strong>, cells were sorted using a Flow Cytometer with a 70 &#181;M nozzle. The maximum collection volume is 1.4 mL.</li>
</ol>
<p class="jove_title">2. Sample Preparation</p>
<p class="jove_content">NOTE: This step is critical. Process the sample very carefully. When removing the supernatant, be very careful not to disturb the cell pellet.</p>
<ol>
	<li>Centrifuge the 1.5 mL tubes containing sorted cells in a swinging bucket rotor at 4 &#176;C, 500 x g, for 5 min. Remove all but approximately 100 &#181;L of the supernatant from the cell pellet very gently using a pipette and a 20 &#181;L pipet tip. Do not disturb the pellet.
	<ol>
		<li>If the sample contains fewer than 5,000 cells, and the total volume of the sorted sample is less than 0.2 mL, transfer the cells first to low binding 0.2 mL PCR tubes before centrifugation.</li>
		<li>If sample contains fewer than 5,000 cells and volume is more than 0.2 mL, spin the cells down with a centrifuge at 4 &#176;C, 500 x g, for 5 min, and remove all but 200 &#181;L of the supernatant. Re-suspend the pelleted cells in the remaining 200 &#181;L of supernatant, then transfer the cells to the 0.2 mL PCR tubes and spin down again. Remove all but 20 - 30 &#181;L from the pellet after the second spin and re-suspend cells.</li>
	</ol>
	</li>
	<li>Resuspend the cells in the supernatant left in the tube. If necessary,add additional PBS in 2% FBS/PBS (you can also use 2% bovine serum albumin (BSA)/PBS, or PBS alone) to obtain a concentration 5 x10<sup>4</sup> to 5 x 10<sup>5</sup> cells/mL.<br />
	NOTE: Use the cell counts collected by cytometry to estimate the volume used to suspend cells.</li>
	<li>Use 2 - 5 &#181;L of the re-suspended cells to determine an accurate cell concentration using an automated cell counter (following the manufacturer&#39;s instructions) or a hemocytometer<sup class="xref">9</sup>.</li>
	<li>Transfer a volume of re-suspended cells containing the desired number of cells to new 1.5 mL tubes. For the experiment in <strong class="xfig">Figure 1</strong>, 2,000, 1,000, or 500 HSCs or HPs were transferred. The desired number of cells depends on the strength of the signal obtained by Western blot, and is determined empirically. Perform the transfer using a 20 &#181;L or 100 &#181;L Eppendorf pipette tip.</li>
	<li>Centrifuge the cells in a swinging bucket rotor at 4 &#176;C, 500 x g, for 5 min.</li>
	<li>To generate 100x stock solutions, dissolve proteasome and phosphatase inhibitors in DMSO according to the manufacturer&#39;s instructions.</li>
	<li>Prepare 2x Laemmli sample buffer by diluting the 4x Laemmli sample buffer provided by the manufacturer with an equivalent amount of distilled water. Add an appropriate amount of the 100x stock of proteasome and phosphatase inhibitors to obtain a final concentration of 2x inhibitors in 2x Laemmli sample buffer.<br />
	NOTE: The 2x Laemmli sample buffer can be made in advance, but the proteasome and phosphatase inhibitors should be added immediately before adding the 2x Laemmli sample buffer (plus inhibitors) to the cell pellet to lyse the cells, as described in the following step.</li>
	<li>Carefully remove a portion of the supernatant from the cell pellet, and to the supernatant remaining in the tube with the cell pellet, add an equal volume of 2x Laemmli sample buffer plus proteasome and phosphatase inhibitors to achieve a final concentration of 500 cells/&#181;L in 1x Laemmli sample buffer.<br />
	NOTE: For example, if you transfer 2,000 cells in a total volume of 20 &#181;L to a tube in Step 2.5, after centrifuging the cells (Step 2.6) you should remove 18 &#181;L of the supernatant from the pellet, and to the 2 &#181;L of supernatant that is remaining add an equal volume (2 &#181;L) of 2x Laemmli sample buffer to achieve a final concentration of 500 cells/&#181;L in 1x Laemmli sample buffer.</li>
	<li>Resuspend the pellet to generate the lysate as soon as possible. At this point, the samples can be immediately electrophoresed through SDS-PAGE gels, or can be snap frozen in liquid N<sub>2</sub> and stored at -80 &#176;C for future use.</li>
</ol>
<p class="jove_title">3. Electrophoresis</p>
<ol>
	<li>Heat the lysates in a heat block at 95 &#176;C or in boiling water for 5 min.</li>
	<li>Electrophorese 1 - 40 &#181;L of the lysates (containing from 500 up to 20,000 cell equivalents) through SDS-PAGE gels at 100V using standard protocols<sup class="xref">10</sup>.<br />
	NOTE: The volume of lysate loaded into the gel is determined by the number of cells needed to generate the desirable signal, and will depend on the abundance of the protein of interest, and the quality of the antibody. The data in <strong class="xfig">Figure 1</strong> were obtained with 2,000, 1,000, and 500 cell equivalents of lysate. The width of the well should be in the range of 3 mm to 1.5 mm. 1.5 mm teeth can be cut from a commercially available comb with wider teeth using scissors.</li>
</ol>
<p class="jove_title">4. Transfer and Block</p>
<p class="jove_content">NOTE: Perform a Western blot following standard protocols<sup class="xref">11</sup>. The steps are briefly outlined here:</p>
<ol>
	<li>Pretreat a polyvinylidene difluoride (PVDF) membrane with methanol for 10 s, then wash the membrane briefly with distilled water.</li>
	<li>Transfer the protein from the SDS-PAGE gel to the membrane following the instructions in the manual provided by the manufacturer of the transfer apparatus.</li>
	<li>Following transfer, block the membrane with 2 mL of 5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween) overnight at 4 &#176;C following standard protocols<sup class="xref">11</sup>.</li>
</ol>
<p class="jove_title">5. Antibody Labeling</p>
<ol>
	<li>Incubate the membrane with antibodies on a shaker overnight at 4 &#176;C using antibody dilutions recommended by the vendor.<br />
	NOTE: In the Table of Materials, antibodies, which we have validated for HSCs and HPs and the corresponding antibody dilutions, are shown. Perform the antibody labeling using standard procedures<sup class="xref">8</sup>.</li>
</ol>
<p class="jove_title">6. Detection</p>
<ol>
	<li>Wash the membrane 3 times, 5 min for each wash in PBST at room temperature.</li>
	<li>Incubate the membrane with 2 mL of enhanced chemiluminescence buffer for 1 min at room temperature. Detect the signals using an imaging system following the manufacturer&#39;s instructions, or autoradiography film and film processor.</li>
</ol>

<ol>
	<li>Krutzik, P. O., Clutter, M. R., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+analysis+of+murine+immune+cell+surface+markers+and+intracellular+phosphoproteins+by+flow+cytometry.">Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry.</a> <em style='font-style: italic'>J Immunol</em>. <b>175</b> (4), 2357-2365 (2005).</li><li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+profiling+at+the+single-cell+level+identifies+a+distinct+signaling+signature+in+murine+hematopoietic+stem+cells.">Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.</a> <em style='font-style: italic'>Stem Cells</em>. <b>30</b> (7), 1447-1454 (2012).</li><li>Signer, R. A., Magee, J. A., Salic, A., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+require+a+highly+regulated+protein+synthesis+rate.">Haematopoietic stem cells require a highly regulated protein synthesis rate.</a> <em style='font-style: italic'>Nature</em>. <b>509</b> (7498), 49-54 (2014).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+differentiation+checkpoint+limits+hematopoietic+stem+cell+self-renewal+in+response+to+DNA+damage.">A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage.</a> <em style='font-style: italic'>Cell</em>. <b>148</b> (5), 1001-1014 (2012).</li><li>Hoshii, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+is+essential+for+leukemia+propagation+but+not+stem+cell+self-renewal.">mTORC1 is essential for leukemia propagation but not stem cell self-renewal.</a> <em style='font-style: italic'>J Clin Invest</em>. <b>122</b> (6), 2114-2129 (2012).</li><li>Signer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rate+of+protein+synthesis+in+hematopoietic+stem+cells+is+limited+partly+by+4E-BPs.">The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.</a> <em style='font-style: italic'>Genes Dev</em>. <b>30</b> (15), 1698-1703 (2016).</li><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx1+deficiency+decreases+ribosome+biogenesis+and+confers+stress+resistance+to+hematopoietic+stem+and+progenitor+cells.">Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells.</a> <em style='font-style: italic'>Cell Stem Cell</em>. , (2015).</li><li>Loven, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+global+gene+expression+analysis.">Revisiting global gene expression analysis.</a> <em style='font-style: italic'>Cell</em>. <b>151</b> (3), 476-482 (2012).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemoacytometer+to+Count+Cells.">Using a Hemoacytometer to Count Cells.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Protein+with+SDS-PAGE.">Separating Protein with SDS-PAGE.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> <em style='font-style: italic'>Basic Methods in Cellular and Molecular Biology</em>. , (2017).</li><li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> <em style='font-style: italic'>Nat Rev Mol Cell Biol</em>. <b>11</b> (2), 113-127 (2010).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> <em style='font-style: italic'>J Vis Exp</em>.  (93), e52099 (2014).</li></ol>

<p class="jove_content">The authors have no conflict of interests to declare.</p>

<p class="jove_content">Western Blotting is a common technique for detecting specific proteins and the activation of signaling pathways in tissues or cells. By introducing small adjustments to a commonly used procedure, we were able to routinely detect 15 different proteins (<strong>Table of Materials</strong>) in 15,000 HSCs, and in some cases in as few as 500 HSCs. The most critical steps in this protocol are: 1) accurately counting the cells, 2) minimizing the number of transfers between tubes, and 3) lysing the cells directly with Laemmli sample buffer. When lysing the cell pellet with Laemmli sample buffer, we found that rather than removing all of the supernatant from the cell pellet and re-suspending it in Laemmli Sample Buffer, if we instead left a small volume of supernatant in the tube and added an equivalent volume of 2x Laemmli Sample Buffer, we could avoid cell loss. Centrifuging the cells in a swinging bucket rotor also decreased the risk of cell loss. Reducing the width of the well by trimming the comb teeth improved the sensitivity of detection.</p>
<p class="jove_content">With these simple adjustments to the procedure, we were able to obtain reproducible results equivalent to those in published papers that had used 10 - 20 times more cells<sup class="xref">3</sup><sup>,</sup><sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup><sup>,</sup><sup class="xref">6</sup><sup>,</sup><sup class="xref">7</sup>. Further, the blots can be stripped for re-blotting following standard procedures<sup class="xref">13</sup>, increasing the amount of information that can be obtained from a small number of cells. The ability to detect proteins of interest will be limited by the quality of the antibody and the protein abundance. This modified technique should greatly reduce the number of animals necessary to obtain protein data from rare cell populations.</p>

<p class="jove_content">Hematopoietic stem cells (HSCs) are self-renewing cells that can give rise to all blood lineages. They are relatively rare cells in the bone marrow, rendering biochemical analyses difficult. Approaches suitable for analyzing rare cells, such as flow cytometry, have been extremely useful for quantifying relative amounts of cell surface markers and intracellular proteins. However, the analysis of intracellular proteins necessitates the use of cell permeabilization procedures to enable antibody access, and not all cell surface epitopes survive these procedures<sup class="xref">1</sup><sup>,</sup><sup class="xref">2</sup>. In addition, antibodies that discriminate between different protein isoforms or cleavage products are not often available for flow cytometry, and therefore investigators still rely on Western blots for certain types of analyses.</p>
<p class="jove_content">Western blot analysis of cell lysates is a routine procedure in most laboratories. Cells can be purified under native conditions that preserve the epitopes of cell surface molecules, and cell lysates can subsequently be prepared and analyzed. However, the analysis of proteins in rare primary cell populations by Western blot can require euthanizing large numbers of animals to obtain enough cells. By making small adjustments to several steps, a conventional Western blotting protocol was able to detect proteins in relatively small numbers of HSCs (500 - 15,000, depending on the protein of interest). The adjustments include accurately counting the cells, carefully handling the cell pellet, reducing transfers of cells between tubes to minimize cell loss, and lysing a defined number of cells with a concentrated loading buffer containing proteasome and phosphatase inhibitors. Many published reports include Western blots obtained with 20,000 or more HSCs<sup class="xref">3</sup><sup>,</sup><sup class="xref">4</sup><sup>,</sup><sup class="xref">5</sup><sup>,</sup><sup class="xref">6</sup><sup>,</sup><sup class="xref">7</sup>; this simple procedure will reduce the number of cells and experimental animals required to produce equivalent data by between 4 and 40 fold. The protocol is designed to normalize results on a per cell basis, rather than to an internal control. This enables detection of overall reductions in protein levels that can be overlooked if data are normalized to an internal control. The importance of normalizing on a per cell basis was described for the analysis of gene expression data<sup class="xref">8</sup>, and the same principle applies to quantifying proteins by Western blot. This optimized protocol should be useful for anyone needing to analyze small numbers of cells.</p>

<table><tbody><tr><td>sodium dodecyl sulfate (SDS)</td><td>Fisher Scientific</td><td>BP166-500</td><td>SDS-PAGE</td></tr><tr><td>TEMED</td><td>Fisher Scientific</td><td>BP150-20</td><td>SDS-PAGE</td></tr><tr><td>30% Acrylamidel Bis solution</td><td>Bio-Rad</td><td>1610158</td><td>SDS-PAGE</td></tr><tr><td>1.5M Tris-HCl pH8.8</td><td>TEKNOVA</td><td>T1588</td><td>SDS-PAGE</td></tr><tr><td>1.0M Tris-HCl pH6.8</td><td>TEKNOVA</td><td>T1068</td><td>SDS-PAGE</td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>BP179-25</td><td>SDS-PAGE</td></tr><tr><td>mini-protean 3 comb</td><td>Bio-Rad</td><td>1653360</td><td>SDS-PAGE</td></tr><tr><td>Mini-PROTEAN Tetra Cell</td><td>Bio-Rad</td><td>1658005EDU</td><td>SDS-PAGE</td></tr><tr><td>Transfer System</td><td>Bio-Rad</td><td>1704155EDU</td><td>transfer</td></tr><tr><td>PowerPac HC Power Supply</td><td>Bio-Rad</td><td>1645052EDU</td><td>electrophoresis</td></tr><tr><td>Imaging System</td><td>Bio-Rad</td><td>1708195</td><td>signal detection</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747EDU</td><td>sample preparation</td></tr><tr><td>10x Tris/Glycine/SDS Electrophoresis Buffer</td><td>Bio-Rad</td><td>1610732EDU</td><td>electrophoresis</td></tr><tr><td>2-Mercaptoethanol</td><td>Bio-Rad</td><td>1610710EDU</td><td>sample preparation</td></tr><tr><td>RTA Mini PVDF Transfer Kit, for 40 blots</td><td>Bio-Rad</td><td>1704272</td><td>transfer</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma</td><td>11697498001</td><td>sample preparation</td></tr><tr><td>Phosphatase Inhibitor Cocktailrs</td><td>Gold Biotechnology</td><td>GB-450-1</td><td>sample preparation</td></tr><tr><td>EIF4G</td><td>Cell signaling</td><td>2469</td><td>WB antibody, validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>p-Rps6</td><td>Cell signaling</td><td>4858</td><td>WB antibody,validated in 1000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): Fig2</td></tr><tr><td>actin(HRP conjugated)</td><td>abcam</td><td>ab49900</td><td>WB antibody,validated in 500 cells&lt;br/&gt; antibody concentration: 1:5000&lt;br/&gt; reference(figure): Fig1, Fig2</td></tr><tr><td>LC-3</td><td>Abcam</td><td>ab48394</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>S6</td><td>Cell Signaling</td><td>2317</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>4EBP1</td><td>Cell Signaling</td><td>9644</td><td>WB antibody, validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>p-4EBP1</td><td>Cell Signaling</td><td>2855</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>TSC2</td><td>Cell Signaling</td><td>4308</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>HSP90</td><td>Cell Signaling</td><td>4877</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (Fig5)</td></tr><tr><td>PTEN</td><td>Cell Signaling</td><td>9188</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>mTOR</td><td>Cell Signaling</td><td>2983</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-mTOR</td><td>Cell Signaling</td><td>5536</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>PP2AB</td><td>Cell Signaling</td><td>4953</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>p-AMPKa</td><td>Cell Signaling</td><td>2535</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:1000&lt;br/&gt; reference(figure): ref5 (FigS1)</td></tr><tr><td>actin</td><td>Santa Cruz</td><td>sc-47778</td><td>WB antibody,validated in 15000 cells&lt;br/&gt; antibody concentration: 1:3000&lt;br/&gt; reference(figure): ref5 (Fig4)</td></tr><tr><td>lineage depletion kit (mouse)</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-041-305</td><td>hematopoietic stem and progenitor cells purification</td></tr><tr><td>SCF</td><td>PeproTech</td><td>250-03</td><td>phosphorylation stimulation</td></tr><tr><td>APC-Cy7 c-kit antibody</td><td>ThermoFisher</td><td>A15423</td><td>cell sorting</td></tr><tr><td>anti-B220</td><td>ThermoFisher</td><td>48-0452-82</td><td>cell sorting</td></tr><tr><td>anti-CD3e</td><td>ThermoFisher</td><td>48-0031-82</td><td>cell sorting</td></tr><tr><td>anti-Mac1</td><td>ThermoFisher</td><td>48-0112-82</td><td>cell sorting</td></tr><tr><td>anti-Gr1</td><td>ThermoFisher</td><td>48-5931-82</td><td>cell sorting</td></tr><tr><td>anti-Ter119</td><td>ThermoFisher</td><td>48-5921-82</td><td>cell sorting</td></tr><tr><td>Spacer Plates with 1.0 mm Integrated Spacers</td><td>Bio-Rad</td><td>1653311</td><td>SDS-PAGE</td></tr><tr><td>BSA</td><td>Research Products International</td><td>A30075</td><td>membrane blocking</td></tr><tr><td>serum</td><td>Atlanta Biologicals</td><td>A12450</td><td>medium supplement</td></tr><tr><td>cell counter</td><td>Invitrogen</td><td>AMQAF1000</td><td>cell counting</td></tr><tr><td>hemocytometer</td><td>ThermoFisher</td><td>&amp;nbsp;S17040</td><td>cell counting</td></tr><tr><td>flim</td><td>Denville Scientific</td><td>E3012</td><td>signal detection</td></tr><tr><td>medical film processor</td><td>Konica</td><td>SRC-101A</td><td>signal detection</td></tr><tr><td>Supersignal West Femto Maximum Sensitivity Substrate</td><td>Therom Scientific</td><td>34096</td><td>signal detection</td></tr><tr><td>Allegra X-15R Centrifuge (Rotor SX4750A)</td><td>Beckman Coulter</td><td>X-15R</td><td>sample preparation</td></tr><tr><td>BLUEstain protein ladder</td><td>Gold Biotechnology</td><td>P007-500</td><td>electrophoresis</td></tr><tr><td>cell sorter</td><td>BD</td><td>FACSAria II</td><td>sample preparation</td></tr><tr><td>Incublock</td><td>Denville Scientific</td><td>I0510</td><td>electrophoresis</td></tr></tbody></table>

a western blotting protocol for small numbers of hematopoietic stem cells

<p class="jove_content">Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.</p>

<p class="jove_content" fo:keep-together.within-page="1">Representative results from 500 - 2,000 purified HSCs and HPs are shown in <strong class="xfig">Figure 1</strong> and <strong class="xfig">Figure 2</strong>. The &#946;-actin signal in <strong class="xfig">Figure 1</strong> can be detected from as few as 500 HSCs and HPs purified from the bone marrow of one mouse. Note that loading the lysates into 1.5 mm wells produced a much stronger signal from 500 HPs than loading into 3.0 mm wells. <strong class="xfig">Figure 2</strong> is a Western blot of EIF4G and the phosphorylation of Rps6 (p-Rps6), both of which are involved in the regulation of protein translation<sup class="xref">12</sup>, in HSCs, and in HPs with and without stimulation by stem cell factor (SCF) <em>in vitro</em>.</p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56855/56855fig1.jpg" /><br />
<strong>Figure 1:&#160; Western Blot for &#946;-actin performed with lysates from murine HSCs and HPs.</strong> HSCs were sorted from lineage depleted murine bone marrow cells as Lin<sup>-</sup>Sca1<sup>+</sup>Kit<sup>+</sup> Flt3<sup>-</sup> cells, and HPs were Lin<sup>-</sup>Sca1<sup>-</sup>Kit<sup>+</sup>. Lysates prepared from different numbers of cells were electrophoresed through a 12% SDS-PAGE gel prepared using mini glass plates with 1 mm spacers. The blot was probed with antibody to &#946;-actin. The blot shows the comparative &#946;-actin signals from 500 to 2,000 HPs when the lysates were loaded into 1.5 mm wells (lanes 4 -&#160;6) as compared to 3 mm wells (lanes 1 - 2). Lysates from 2,000 to 500 cells were loaded onto lanes 7 to 9. M, molecular weight markers. <a href="https://cloudflare.jove.com/files/ftp_upload/56855/56855fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>
<p class="jove_content" fo:keep-together.within-page="1"><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56855/56855fig2.jpg" /><br />
<strong>Figure 2: Western Blot analysis of freshly isolated HSCs, and HPs stimulated <em>in vitro</em> with the cytokine Stem Cell Factor (SCF). </strong>HPs were purified by fluorescence activated cell sorting (FACS), centrifuged, resuspended in 2% FBS/PBS, counted, then split into two tubes. The HPs in the first tube were stimulated with SCF (10 ng/mL) for 5 min at 37 &#176;C (+SCF), and HPs in the second tube were cultured for 5 min in the absence of SCF (-SCF). The cells were then centrifuged and the cell pellet lysed with Laemmli sample buffer. HSCs were purified and directly lysed with Laemmli sample buffer without culturing. The lysates were loaded into 1.5 mm wells and electrophoresed through 12% SDS-PAGE gels. The blot was developed with antibodies to elongation initiating factor 4G (EIF4G), &#946;-actin, and phosphorylated small ribosome subunit S6 (p-Rps6). <a href="https://cloudflare.jove.com/files/ftp_upload/56855/56855fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></p>

Watch this Scientific Journal Video about A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells at JoVE.com

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

<p class="jove_content">A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.</p>

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

a-western-blotting-protocol-for-small-numbers-hematopoietic-stem

Nanyang Technological University

Research

JoVE Journal

Biochemistry

10.3K Views.  Cincinnati Children's Hospital Medical Center. A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Video: A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Buffers

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Preparation of Solutions<button class="expand">Expand</button>
	<p><em>Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.</em></p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">To set up for this lab experiment, wear the appropriate personal protective equipment, including a lab coat, chemical splash goggles, and gloves. All solutions for this lab should be prepared in a chemical fume hood.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Prepare 100 mL of a 1 M NaOH solution. <strong>Note: </strong>NaOH is strongly corrosive, so use caution while handling it.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Measure 4 g of solid NaOH and pour it in a 125-mL Erlenmeyer flask. Then, add 50 mL of deionized water to the flask. Stir the mixture on a stir plate to dissolve the NaOH.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Label a 100-mL polyethylene bottle as &#39;1 M NaOH&#39;. Once the NaOH has dissolved and the solution appears homogeneous, add another 50 mL of deionized water. Then, retrieve the stir bar and transfer the solution to the labeled bottle. Place the capped bottle in the back of the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Prepare 10 mL of a 0.56 mM solution of neutral red in deionized water. Measure 1.6172 mg of neutral red into a 10-mL volumetric flask.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Use a pipette to fill the flask about 2/3 full with deionized water. Cover the flask with plastic paraffin film and agitate the mixture until the neutral red dissolves. Then, remove the film and fill the flask to the marked line. Place a micro-stir bar in the flask and stir the solution until it appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Label a 20-mL vial as &#39;0.56 mM neutral red&#39; and transfer the solution to the vial. Cap the vial and store it in the fridge.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare 10 mL of a 0.66 mM solution of riboflavin-binding protein in the same way, starting by weighing 198 mg of the protein. Transfer the solution to a labeled 20-mL vial and store the solution in the fridge.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Prepare or obtain a set of buffers. Here, we will go through the preparation of 100 mL of 50 mM pH 6.5 monosodium phosphate buffer, adjusted with 1 M NaOH as an example.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Measure 0.5995 g of anhydrous monosodium phosphate into a 125-mL Erlenmeyer flask. Add 80 mL of deionized water to the flask and begin stirring the mixture.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">While the mixture stirs, turn on and calibrate a digital pH meter.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once the salt has dissolved and the solution appears homogeneous, clamp the pH sensor in the buffer and wait for the reading to stabilize.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Confirm that the 1 M NaOH solution has cooled to room temperature. Then, slowly add NaOH to the stirring buffer solution with pauses to let the pH stabilize until the buffer reaches pH 6.5. This may only take 1 or 2 mL of NaOH. Then, turn off the stir motor, rinse the pH probe, and retrieve the stir bar.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Pour the pH adjusted buffer into a clean 100-mL volumetric flask and bring the solution up to the 100-mL mark with deionized water. Seal the flask with plastic paraffin film and invert it until the solution appears homogeneous.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a clean 125-mL polyethylene bottle as &#39;50 mM monosodium phosphate buffer pH 6.5&#39;. Transfer the solution to the bottle and cap it. Store the buffer in the instructor&#39;s hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Prepare additional buffers in a similar fashion, using 1 M NaOH or 2 M HCl as appropriate to adjust the pH. Alter the buffer concentrations as needed to achieve roughly the same ionic strength.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">When finished, store the 1 M NaOH in a corrosives cabinet, clean the glassware and equipment using your standard procedures, and throw out disposable items in the lab trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Preparation of the Laboratory<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Bring a container of anhydrous monosodium phosphate to the analytical balance area and confirm that there are sufficient supplies of weighing paper, laboratory wipes, and clean spatulas.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Set out the following lab equipment and glassware at each lab station (we suggest that students work in pairs):
		<table align="center" height="224" style="border-collapse: collapse; font-size: 17px; line-height: 24px;">
			<tbody>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Stir plate</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Lab stand with thermometer clamp</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;pH meter</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Handheld spectrophotometer</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Calibration buffers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;400-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;100-mL beakers</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;25-mL beaker</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;50-mL graduated cylinder</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;10-mL graduated cylinders</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Small watch glass</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Magnetic stir bar</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Funnel</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Forceps</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Plastic wash bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;250- or 500-mL capped polyethylene bottle</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Roll of laboratory tape</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;2&#160;&#160;&#160;&#160;Plastic pipettes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Labeling pen</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;Box of laboratory wipes</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;1-mL micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;1&#160;&#160;&#160;&#160;200-&#181;L micropipette with tips</td>
				</tr>
				<tr>
					<td style="border: 1px solid; padding: 0px;">&#160;10&#160;&#160;&#160;&#160;1.5-mL cuvettes with caps</td>
				</tr>
			</tbody>
		</table>
		</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">Set out additional disposable plastic pipettes so that everyone will have easy access to them.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Place labeled bottles or flasks containing the buffers in a central area.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Label a 1-mL micropipette for each buffer and place the micropipettes with the corresponding buffers along with a box of pipette tips.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Portion out the neutral red and protein solutions. Label three 5-mL test tubes as &#39;neutral red&#39; and three 5-mL test tubes as &#39;riboflavin-binding protein&#39;. Then, take the vials of neutral red solution and riboflavin-binding protein from the fridge and place about 2 mL of the neutral red solution and 2 mL of riboflavin-binding protein solution in the appropriately labeled test tubes.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Set out the neutral red and protein solutions so the student groups can share the test tubes.</div>
		</li>
	</ul>
	</li>
</ol>

Buffers: Common Ion Effect, Henderson-Hasselbalch Equation, and Buffer Capacity - Prep

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Preparation of SolutionsExpand
	Here, we show the laboratory preparation for 10 students ...

Education

JoVE Lab Manual

Chemistry

Lab Manual Sub Articles

Thin-Layer Chromatography

<p>Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Effects of Mobile Phase Composition on Compound Separation<button class="expand">Expand</button>
	<p>In this section of the lab, you will use TLC to compare how well pure hexane and a mixture of 60% hexane and 40% ethyl acetate can separate phenol, benzoic acid, and butyl phenyl ether by measuring the distance the compound traveled in each solvent.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-2">
		<div class="lab-step">Before starting the lab, put on a lab coat, safety glasses, and nitrile gloves. Note: Hexane and ethyl acetate are both volatile, toxic, and flammable, so always work with them in a fume hood. Change your gloves if you get solvent on them, as hexane and ethyl acetate will permeate nitrile gloves within minutes.</div>
		</li>
		<li data-sub-note-item="1-3">
		<div class="lab-step">Now, obtain a paper towel and lay it on the benchtop. Carefully pick up a TLC plate using tweezers at the very end of the plate.<strong> Note: </strong>Always hold TLC plates by the edges or at the very top with tweezers to avoid damaging or contaminating the silica gel.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Look for chips or scratches in the silica gel and get a new plate if necessary. It&#39;s fine if there is a little chipping at only one end, since that can be the top.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Place the plate on the paper towel in your fume hood, and lay a metric ruler next to it. Use a graphite pencil to gently make a small mark on the silica gel ~1 cm from the top of the plate. Write &#8216;100&#8217; on the plate above the mark.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Next, gently mark the plate 1 cm above the bottom on both sides. Carefully trace a straight line between the bottom marks to make the starting line.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Then, add 3 evenly spaced small marks to the starting line with the outer marks at least 0.5 cm from the sides of the plate. <strong>Note:</strong> It&#39;s important for the silica gel to be undamaged here, so if you scratch it with the pencil, prepare a new plate.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Now, trace the outline of the TLC plate in your lab notebook and precisely copy the marks on the plate. Label the three marks as &#8216;phenol&#8217;, &#8216;benzoic acid&#8217;, and &#8216;butyl phenyl ether&#8217;. You may also want to label the marks on the plate itself.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Prepare a second TLC plate labeled &#8216;60/40&#8217; in the same way. Now, bring the labeled plates to the common bench and locate the 1 mg/mL solutions of phenol, benzoic acid, and butyl phenyl ether in ethanol.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Dip a clean capillary into one of the three solutions. Briefly and lightly dab the capillary squarely against the intersection between the starting line and the mark for that compound. You should spot twice for each compound.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use the same capillary to spot the same solution on your second TLC plate. The spots should be no more than 2 mm in diameter. Then, discard the capillary and close the vial.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Spot the other two solutions on the plates, in the same way, using clean capillaries. Be careful not to cross-contaminate the solutions. <strong>Note</strong>: If a spot is very light or very small, wait for it to dry, and then spot the same solution on top of the dry spot. If the spot is still wet, capillary action could make it too wide and dilute.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Once you have made three spots on each plate, discard the capillaries and bring the plates back to your hood. Keep the plates lying flat while they dry.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Now, label a 50-mL jar &#8216;100% hexane&#8217; and bring the jar, its cap, and a 10-mL graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Measure 2 &#8211; 3 mL of pure hexane with your graduated cylinder and pour it into the jar to fill it to a depth of 0.62 &#8211; 0.7 cm. Cap the bottle and jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Label a second 50-mL jar &#8216;60% hexane, 40% ethyl acetate&#8217; and bring it and a clean graduated cylinder to the solvent hood. Measure 2 &#8211; 3 mL of the 60/40 mixture into the jar and return to your fume hood.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Stand the plates next to the jars to compare the relative position of the solvent and the spots. The solvent level should be below the spots for each plate.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Then, obtain three pieces of filter paper, put a filter paper upright against the wall in each jar, and then cap the jars tightly.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Initial each plate above the mark so you can identify them later. Confirm that the filter papers in the jars have become saturated with solvent and that the spots on the plates are completely dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, open the 100% hexane jar and use tweezers to gently place the 100 plate in the jar, keeping the plate level with the solvent surface as it enters the solvent. Close the jar tightly, being careful not to jostle the plate.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Leave the jar still as the solvent travels up the plate. Development in pure hexane usually takes about 7 &#8211; 10 min. Keep an eye on the plate to make sure that the solvent doesn&#39;t reach the top.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">When the pure hexane solvent front is about 1 cm from the top of the plate, retrieve it using tweezers. Lay it on the paper towel with the silica gel facing up and quickly mark the solvent front with a line and pencil across the plate. Leave the plate lying flat as the solvent evaporates.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Place the other plate in the 60/40 eluent in the same way. Wait until the solvent front of the 60/40 plate is about 1 cm from the top, and then retrieve it and quickly mark the solvent front in the same way.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Once the 100% hexane plate is completely dry, pick it up with tweezers and bring it and a pencil to the UV lamp on the common bench.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Place the plate under the lamp and illuminate it with UV light. The plate will glow green because of a fluorescent marker in the silica gel layer. The dark spots are the compounds.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Carefully outline every spot in pencil, even if they haven&#39;t moved very far.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Then, turn off the lamp and bring your plate to the iodine development hood. Open the iodine chamber. Use tweezers to gently place the plate upright in the iodine chamber, and then cover the chamber again.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Wait 3 &#8211; 4 min for the iodine vapor to stay in the spots. Then, retrieve the plate with tweezers, close the chamber, and outline the brown and yellow spots on the plate. These spots will fade, so mark them quickly.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Visualize the dry 60/40 plate under UV light and then with iodine vapor in the same way.<strong> Note: </strong>To avoid side reactions, always check plates under UV light before exposing them to iodine.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Now, precisely copy the spots and the solvent fronts onto the corresponding drawings of the plates in your lab notebook. For each plate, measure and record the height of the solvent front relative to the starting line.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Lastly, measure the distance between the center of each spot and its starting line, and record it in your lab notebook. You can now move on to the next part of the lab.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Unknown Compound Identification<button class="expand">Expand</button>
	<p>In the second half of the lab, you&#39;ll use TLC to find out what&#39;s in an unknown analgesic. It could contain one or more of these compounds: aspirin, caffeine, and acetaminophen. These compounds are highly polar, so you&#39;ll use a premade mixture of 90% ethyl acetate and 10% hexane as the eluent.</p>
	<p>You&#39;ll run the three reference compounds and the unknown all on the same plate. Later, you&#39;ll use the Rf values of the reference compounds to identify the unknown. First, double-check which unknown material you were assigned to analyze before starting.</p>
	<ul class="lab-items">
		<li data-sub-note-item="2-3">
		<div class="lab-step">Now, lay a clean paper towel in the hood and obtain a new TLC plate. Prepare the top mark and the starting line the same way as before. Make four evenly spaced ticks on the plate, making sure that the outer marks are at least 0.5 cm away from the edges.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Draw an exact copy of the plate in your lab notebook and label the tick marks as &#8216;aspirin&#8217;, &#8216;acetaminophen&#8217;, &#8216;caffeine&#8217;, and &#8216;unknown&#8217;.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Now, bring your plate to the common bench. Spot solutions of aspirin, acetaminophen, caffeine, and the unknown that you were assigned on the appropriate marks using a clean capillary for each solution. <strong>Note: </strong>When you are spotting the unknown solution, don&#39;t dip the capillary too far into the solution vial to avoid clogging your capillary with insoluble material from the unknown substance.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Discard the capillaries and close the vials before bringing the plate back to your fume hood. Place the plate on a paper towel to finish drying.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Next, label a clean 50-mL screw-top jar &#8216;90% ethyl acetate, 10% hexane&#8217;, and bring it in a clean graduated cylinder to the solvent hood.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Place 2 &#8211; 3 mL of the 90/10 solution in the jar and return to your fume hood. Confirm that the solvent level will be below the spots on the plate. Then, place a clean filter paper upright in the jar and close it tightly.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Once the TLC plate is dry and the filter paper is saturated, carefully place the plate in the solvent. <strong>Note:</strong> It&#39;s essential to hold the plate level when you do this. If the solvent front is diagonal, the solvent distance traveled will be different for each spot, making it impossible to visually compare the unknown to the reference spots.</div>
		</li>
		<li data-sub-note-item="2-10">
		<div class="lab-step">Wait for the solvent front to reach 1 cm from the top of the plate. Then, retrieve the plate, mark it solvent front, and let it dry while lying flat.</div>
		</li>
		<li data-sub-note-item="2-11">
		<div class="lab-step">Once the plate is dry, visualize it with UV light and in the iodine chamber, and outline the spots in pencil.</div>
		</li>
		<li data-sub-note-item="2-12">
		<div class="lab-step">The unknown material could be a mixture of compounds, so look carefully for spots in line with all three reference spots.</div>
		</li>
		<li data-sub-note-item="2-13">
		<div class="lab-step">Then, precisely copy the spots onto the drawing of the plate in your lab notebook. Measure and record the height of the solvent front and the spots relative to the starting line.</div>
		</li>
		<li data-sub-note-item="2-14">
		<div class="lab-step">When you are finished, retrieve the solvent-saturated filter papers from the jars and let them dry in the hood.</div>
		</li>
		<li data-sub-note-item="2-15">
		<div class="lab-step">Pour leftover solvents into the standard organic waste, wash your glassware following your lab&#39;s usual methods, and put away your lab equipment.</div>
		</li>
		<li data-sub-note-item="2-16">
		<div class="lab-step">Throw out used TLC plates in the lab trash or with glass waste, depending on the backing material.</div>
		</li>
		<li data-sub-note-item="2-17">
		<div class="lab-step">Lastly, once the filters are dry, throw them out along with any remaining trash.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="3" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="3-1">
		<div class="lab-step">Calculate the R<sub>f</sub> values for phenol, benzoic acid, and butyl phenyl ether eluted with 100% hexane. For each compound on the TLC plate, measure the distance from the starting line to the center of its spot. Then, divide this value by the distance from the starting line to the solvent front.</div>
		</li>
		<li data-sub-note-item="3-2">
		<div class="lab-step">Only butyl phenyl ether moved a measurable distance from the starting line, which is consistent with it being the least polar. For effective separation, the R<sub>f</sub> values should differ by 0.3 &#8211; 0.7. Thus, we can infer that phenol and benzoic acid are too polar to be separated effectively by an apolar solvent like pure hexane.</div>
		</li>
		<li data-sub-note-item="3-3">
		<div class="lab-step">Calculate the R<sub>f</sub> values for the plate that you ran in a 60/40 mixture of hexane and ethyl acetate. Ethyl acetate is a moderately polar solvent, so the compounds traveled farther and are spaced farther apart on the plate.</div>
		</li>
		<li data-sub-note-item="3-4">
		<div class="lab-step">The separation order shows that benzoic acid, the most polar compound, had the highest affinity for silica gel, whereas butyl phenyl ether, the least polar compound, had the lowest affinity for silica gel. This is consistent with the assumption that less polar compounds travel farther on polar stationary phases.</div>
		</li>
		<li data-sub-note-item="3-5">
		<div class="lab-step">Look at your third plate and calculate the R<sub>f</sub> values for aspirin, acetaminophen, and caffeine. Caffeine, the most polar complex of the three, traveled the least. Acetaminophen traveled the second farthest, while aspirin, the least polar, traveled the most.</div>
		</li>
		<li data-sub-note-item="3-6">
		<div class="lab-step">Finally, identify the compounds in your unknown material by comparing its spot or spots to the three reference spots.</div>
		</li>
	</ul>
	</li>
</ol>

Thin-Layer Chromatography: Experimental Setup and Separation - Procedure

Source: Lara Al Hariri and&#160;Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Effects of Mobile Phase Composition on Compound ...

Concentration Dependence

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Concentration Dependence<button class="expand">Expand</button>
	<p>In this experiment, you will combine varying concentrations of aqueous HCl and sodium thiosulfate to make solid sulfur, which rapidly gathers into opaque yellow particles at a certain sulfur concentration. Since the reaction solution starts clear and colorless, you can easily tell when it reaches that concentration.</p>
	<p>You&#39;ll use the same test tube and total volume each time, so it will take the same amount of sulfur to make each solution completely opaque. Thus, you&#39;ll measure the reaction progress by timing how long that takes. You&#39;ll then use that data to estimate the reaction orders of the individual reactants and of the overall reaction.</p>
	<p>Before starting the lab, make a table listing the reactant concentrations, the time to solution opacity, and the solution temperature for the trials.</p>
	<h4><strong>Table 1: Reaction progress</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td><strong>Trial</strong></td>
				<td><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] (M)</strong></td>
				<td><strong>[HCl] (M)</strong></td>
				<td><strong>Time (s)</strong></td>
				<td><strong>Temp (&#176;C)</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">2</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">3</td>
				<td style="text-align: center;">0.2 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">4</td>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">5</td>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">3.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">6</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">6.0 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">7</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">4.5 M</td>
				<td></td>
				<td></td>
			</tr>
			<tr>
				<td style="text-align: center;">8</td>
				<td style="text-align: center;">0.1 M</td>
				<td style="text-align: center;">1.5 M</td>
				<td></td>
				<td></td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_1._Reaction_progress.xlsx" target="_blank">Click Here to download Table 1</a></p>
	<p>We want to make sure that the reactions are all taking place at room temperature. You&#39;ll perform two benchmark trials, three trials with different sodium thiosulfate concentrations, and three trials with different HCl concentrations. You will vary the concentrations by diluting stock solutions of sodium thiosulfate and HCl, as shown in the following tables.</p>
	<h4><strong>Table 2: Sodium thiosulfate dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 0.2 M sodium thiosulfate</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">0.05 M</td>
				<td style="text-align: center;">5 mL</td>
				<td style="text-align: center;">15 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.10 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">10 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.15 M</td>
				<td style="text-align: center;">15 mL</td>
				<td style="text-align: center;">5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">0.20 M</td>
				<td style="text-align: center;">20 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_2._Sodium_thiosulfate_dilutions.xlsx" target="_blank">Click Here to download Table 2</a></p>
	<h4><strong>Table 3: HCl Dilutions</strong></h4>
	<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<tbody>
			<tr>
				<td style="text-align: center;"><strong>Target Concentration</strong></td>
				<td style="text-align: center;"><strong>Volume of 6.0 M HCl</strong></td>
				<td style="text-align: center;"><strong>Volume of DI water</strong></td>
			</tr>
			<tr>
				<td style="text-align: center;">1.5 M</td>
				<td style="text-align: center;">2.5 mL</td>
				<td style="text-align: center;">7.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">3.0 M</td>
				<td style="text-align: center;">5.0 mL</td>
				<td style="text-align: center;">5.0 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">4.5 M</td>
				<td style="text-align: center;">7.5 mL</td>
				<td style="text-align: center;">2.5 mL</td>
			</tr>
			<tr>
				<td style="text-align: center;">6.0 M</td>
				<td style="text-align: center;">10 mL</td>
				<td style="text-align: center;">0 mL</td>
			</tr>
		</tbody>
	</table>
	<p><a href="https://www.jove.com/CDNSource/lm/labs/50/Table_3._HCl_Dilutions.xlsx" target="_blank">Click Here to download Table 3</a></p>
	<p>Remember to use caution when handling HCl, which is toxic and highly corrosive. Sulfur dioxide, which is a gaseous product of this reaction, is also toxic. You will leave the reaction waste in the fume hood overnight to let the sulfur dioxide escape harmlessly.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-6">
		<div class="lab-step">First, put on a lab coat, splash-proof safety glasses, and nitrile gloves.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Fill a plastic wash bottle with deionized water and get a test tube marked with an X.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Clamp the test tube over the center of a small stir plate. The label must be on the opposite side from you. Make sure that you can clearly see the X. Place a small magnetic stir bar in the test tube.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Use a thermometer clamp to secure a glass thermometer in the lower third of the tube, keeping it clear of the X and the stir bar.</div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Cut a sheet of about 20 squares of plastic paraffin film from a roll and bring the sheet back to your hood.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Lay paper towels in the fume hood as a clean surface for labware to be reused.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Label a 600-mL beaker as &#8216;waste&#8217; for the reaction, a 100-mL beaker as &#8216;0.2 M sodium thiosulfate&#8217;, and a 50-mL beaker as &#8216;6 M HCl&#8217;.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Fill the 100-mL beaker with 0.2 M sodium thiosulfate from the stock bottle in the dispensing hood.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">After that, bring the 50-mL beaker and a watch glass to the acid-dispensing hood to obtain 50 mL of 6 M HCl. Cover the filled beaker with the watch glass and bring your portion of HCl back to your hood.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Prepare 20 mL of 0.1 M sodium thiosulfate for the first benchmark trial. Use a 10-mL volumetric pipette to measure 10 mL of 0.2 M sodium thiosulfate and dispense it into a 20-mL volumetric flask. Fill the 20-mL volumetric flask with deionized water to the mark.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Then, cut a square of plastic paraffin film, seal the flask, and invert it several times to thoroughly mix the solution.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Remove the film and pour the solution into the test tube.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">Prepare 10 mL of 3 M HCl. Pipette 5 mL of 6 M HCl into a 10-mL graduated cylinder. Add deionized water to fill the graduated cylinder to the 10-mL line.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Seal the top of the graduated cylinder with plastic paraffin film and thoroughly mix the HCl solution by inverting it several times.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Now, start the stir motor and get a reset stopwatch. Confirm that you can see the X through the solution in the test tube.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Simultaneously start the stopwatch and pour the 3 M HCl into the test tube.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">Watch the X through the solution, which will start becoming cloudy when enough sulfur has been produced to saturate it. Stop the stopwatch exactly when the solution becomes so opaque that the X disappears.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Record the time in your lab notebook for benchmark trial 1 and then reset the stopwatch. Also, record the temperature of the solution.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Now, turn off the stir motor, remove the thermometer, and rinse it with deionized water into the waste beaker.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">Then, empty the test tube into the waste beaker, retrieve the stir bar with long forceps, and rinse the test tube and stir bar with deionized water.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Thoroughly clean the test tube with a test tube brush. Briefly rinse the volumetric flask and graduated cylinder with deionized water as well. Dry everything with paper towels.</div>
		</li>
		<li data-sub-note-item="1-27">
		<div class="lab-step">Repeat the trial in the exact same way for the second benchmark time. If the time is not within 3 - 5 s of the first trial, repeat the benchmark trial until you have two consistent trials.</div>
		</li>
		<li data-sub-note-item="1-28">
		<div class="lab-step">Follow the same overall procedure for each subsequent trial, changing the dilutions as indicated in your table and cleaning your glassware between each trial. Use a 5-mL volumetric pipette as needed when measuring the sodium thiosulfate stock solution in trials four and five.</div>
		</li>
		<li data-sub-note-item="1-29">
		<div class="lab-step">Once you have performed all eight trials, rinse your glassware and tools one last time and neutralize the reaction waste with baking soda. Use caution because sulfur dioxide is one of the reaction products.</div>
		</li>
		<li data-sub-note-item="1-30">
		<div class="lab-step">Transfer the neutralized reaction waste to the designated waste container. Then, pour any remaining sodium thiosulfate solution into its labeled waste container and rinse the beaker with deionized water.</div>
		</li>
		<li data-sub-note-item="1-31">
		<div class="lab-step">Neutralize any remaining HCl with baking soda before flushing it down the drain with tap water.</div>
		</li>
		<li data-sub-note-item="1-32">
		<div class="lab-step">Finally, clean and put away your labware according to your standard practices and throw out any trash remaining in your hood. Your instructor will dispose of the reaction waste later.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">Now, estimate the reaction order of sodium thiosulfate and HCl. Calculate the starting concentration of sodium thiosulfate in the reaction mixture for trials 1 through 5. Looking at the concentration of sodium thiosulfate and the reaction times, we see that as the concentration doubles, the time to opacity is halved.
		<h4><strong>Table 4: Estimating reaction order sodium thiosulfate</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] added</strong></td>
					<td style="text-align: center;"><strong>Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub> volume (mL)</strong></td>
					<td style="text-align: center;"><strong>Total volume (mL)</strong></td>
					<td style="text-align: center;"><strong>[Na<sub>2</sub>S<sub>2</sub>O<sub>3</sub>] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
					<td style="text-align: center;"><strong>Inverse time (s<sup>-1</sup>)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_4._Estimating_reaction_order_sodium_thiosulfate.xlsx" target="_blank">Click Here to download Table 4</a></div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Remember that each trial used the same volume, so it took the same amount of sulfur to make each solution opaque. Thus, the reaction is twice as fast when the sodium thiosulfate concentration is doubled.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">When the concentration increases by a factor of 4, the reaction rate also increases by a factor of 4. This one-to-one relationship suggests that the reaction was first order for sodium thiosulfate. If so, we would expect a plot of rate versus sodium thiosulfate concentration to be linear.</div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">We didn&#39;t measure how much sulfur it took to turn the solution opaque, but we assume that it was the same for each trial. This lets us use the inverse of time to opacity as a stand-in for the rate. The slope won&#39;t be equal to k, but that&#39;s OK for this lab.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Plot inverse reaction time as a function of sodium thiosulfate concentration. This confirms the linear relationship between sulfur production and thiosulfate concentration. Thus, the reaction seems to be first order with respect to thiosulfate.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Use the same method to estimate the reaction order of HCl. First, calculate the starting concentration of HCl in the benchmark trials and in trials 6 through 8.
		<h4><strong>Table 5: Estimating reaction order HCl</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>[HCl] added</strong></td>
					<td style="text-align: center;"><strong>HCl volume (mL) </strong></td>
					<td style="text-align: center;"><strong>Total volume (mL) </strong></td>
					<td style="text-align: center;"><strong>[HCl] in mixture </strong></td>
					<td style="text-align: center;"><strong>Time (s)</strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">1, 2</td>
					<td style="text-align: center;">0.1</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">3</td>
					<td style="text-align: center;">0.2</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">4</td>
					<td style="text-align: center;">0.15</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
				<tr>
					<td style="text-align: center;">5</td>
					<td style="text-align: center;">0.05</td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
					<td style="text-align: center;"></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/50/Table_5._Estimating_reaction_order_HCl.xlsx" target="_blank">Click Here to download Table 5</a></div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Then, look at the concentration of HCl and the reaction times for these trials. The reaction times are nearly identical, so the HCl concentration has no effect on the reaction rate. This means that HCl seems to be zeroth order for this reaction.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Lastly, add up the reaction orders of the reactants to get the overall reaction order.</div>
		</li>
	</ul>
	</li>
</ol>

Concentration Dependence and Reaction Order - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Concentration DependenceExpand
	In this experiment, you will combine varying concentrations of ...

Galvanic Cells

Galvanic Cells: Construction and Reduction Potential Measurement - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Constructing a Lead-Copper Galvanic CellExpand
	In this experiment, you will construct a ...

Electrolytic Cells

<p>Source: Smaa Koraym at Johns Hopkins University, MD, USA</p>
<ol class="note-items">
	<li data-note-item="1" data-parent-collapse="">Electrolytic Cells<button class="expand">Expand</button>
	<p>In this experiment, you will be assembling an electrolytic cell to perform electroplating. An electrolytic cell contains an anode and cathode connected by a power source, which drives the nonspontaneous redox reaction in the cell. When the anode material is oxidized, the ions travel to the cathode, where they are reduced and plated.</p>
	<p>In the case of this experiment, the electrolytic cell contains a copper anode and a brass key for the cathode. Oxidation of the anode creates copper(2+) ions, which are then reduced on the brass key, forming a thin plating of solid copper.</p>
	<ul class="lab-items">
		<li data-sub-note-item="1-3">
		<div class="lab-step">Put on gloves, safety goggles, and a lab apron, which must be worn at all times.</div>
		</li>
		<li data-sub-note-item="1-4">
		<div class="lab-step">Obtain one 10-ohm resistor, one copper sheet electrode, one copper wire, and one piece of emery paper.</div>
		</li>
		<li data-sub-note-item="1-5">
		<div class="lab-step">Use the emery paper to polish the brass key and the copper sheet. Rinse the key and the copper electrode with water. The brass key will be the cathode, and the copper sheet will be the anode.</div>
		</li>
		<li data-sub-note-item="1-6">
		<div class="lab-step">Take one 50-mL beaker and a watch glass to the instructor&#39;s hood and obtain approximately 20 mL of acetone. Place the watch glass on top of the beaker to prevent evaporation and bring it back to your bench.</div>
		</li>
		<li data-sub-note-item="1-7">
		<div class="lab-step">Label the other 50-mL beaker as &#39;organic waste&#39;.</div>
		</li>
		<li data-sub-note-item="1-8">
		<div class="lab-step">Use a disposable pipette to rinse the copper electrode and key with acetone. Collect excess in the waste beaker. Place the key and copper electrode on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-9">
		<div class="lab-step">Measure the initial masses of the key and copper electrode and record their values in your lab notebook.
		<h4><strong>Table 1: Moles of Cu Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td></td>
					<td style="text-align: center;"><strong>Mass of key (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu plated </strong></td>
					<td style="text-align: center;"><strong>Mass of Cu electrode (g) </strong></td>
					<td style="text-align: center;"><strong>Change in mass (g) </strong></td>
					<td style="text-align: center;"><strong>Moles Cu lost </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">0 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_1._Moles_of_Cu_Transferred.xlsx" target="_blank">Click Here to download Table 1</a></div>
		</li>
		<li data-sub-note-item="1-10">
		<div class="lab-step">Set up the electrolytic cell. Use one electrical wire with alligator clips to connect the negative terminal on the battery to the current probe. Wrap the 10-ohm resistor around the other end of the current probe.</div>
		</li>
		<li data-sub-note-item="1-11">
		<div class="lab-step">Use another wire to connect the positive terminal on the battery to the copper electrode.</div>
		</li>
		<li data-sub-note-item="1-12">
		<div class="lab-step">Wrap the copper wire around the brass key and use an electrical wire to connect the resistor on the current probe to the copper wire on the brass key.</div>
		</li>
		<li data-sub-note-item="1-13">
		<div class="lab-step">Obtain about 200 mL of concentrated copper sulfate in a 250-mL beaker. The amount does not need to be exact. Bring the beaker back to your bench and set it on the stir plate. Carefully, place a magnetic stir bar in the beaker and turn the stir setting on to high.</div>
		</li>
		<li data-sub-note-item="1-14">
		<div class="lab-step">Connect the data acquisition system to the current probe and allow the data acquisition system to sense the probe. It should return a reading in amps. Change the rate to two scans per min and the duration to 5 min.</div>
		</li>
		<li data-sub-note-item="1-15">
		<div class="lab-step">Place the brass key in the beaker. Hang it off the side of the beaker so that the key is almost completely submerged but does not touch the magnetic stir bar. <strong>Note: </strong>The copper wire should not be in the solution.</div>
		</li>
		<li data-sub-note-item="1-16">
		<div class="lab-step">Place the copper electrode in the beaker, bending it so that it hangs over the edge of the beaker. Ensure that it is not touching the stir bar or the brass key.</div>
		</li>
		<li data-sub-note-item="1-17">
		<div class="lab-step">Start the scan and let it collect data for 5 min. This will act as a test run to make sure plating occurs on the brass key.</div>
		</li>
		<li data-sub-note-item="1-18">
		<div class="lab-step">After the scan ends, remove the key and copper electrode. The copper plating on the brass key should be evident. If it is not visible, check your circuit to make sure that the battery is connected in the correct orientation.</div>
		</li>
		<li data-sub-note-item="1-19">
		<div class="lab-step">Once you have completed a successful 5-min plating, disconnect the key and copper electrode from the wiring. Remove the copper wire from the key and rinse the key and copper electrode with water and then acetone. Lay them on a paper towel to dry.</div>
		</li>
		<li data-sub-note-item="1-20">
		<div class="lab-step">Measure the masses of the key and the copper electrode and record them in your lab notebook.</div>
		</li>
		<li data-sub-note-item="1-21">
		<div class="lab-step">Reconnect the key and copper electrode and place them back in the solution like before. Restart the run on the data acquisition system, making sure that the rate is set to two scans per min. With the duration set to 30 min, allow the electroplating process to run.</div>
		</li>
		<li data-sub-note-item="1-22">
		<div class="lab-step">After the scan is complete, remove the electrode and key from the solution, disconnect them from the wires, and rinse them with water and then acetone. Allow them to dry on a paper towel, then weigh them and record their masses in your notebook.</div>
		</li>
		<li data-sub-note-item="1-23">
		<div class="lab-step">Reassemble the setup and repeat the electroplating for a second 30-min trial. When the scan is complete, remove the key and copper electrode, rinse them like before, and record their mass.</div>
		</li>
		<li data-sub-note-item="1-24">
		<div class="lab-step">Save the recorded data as a text file on a flash drive.</div>
		</li>
		<li data-sub-note-item="1-25">
		<div class="lab-step">To clean up, turn the stir setting off and remove the stir bar from the solution using forceps. Dispose of the acetone in the organic waste container provided by your instructor. The copper sulfate solution can be reused for future labs, so pour the concentrated copper sulfate back into the carboy.</div>
		</li>
		<li data-sub-note-item="1-26">
		<div class="lab-step">Rinse all of your glassware with water and return it to the instructor.</div>
		</li>
	</ul>
	</li>
	<li data-note-item="2" data-parent-collapse="">Results<button class="expand">Expand</button>
	<ul class="lab-items">
		<li data-sub-note-item="2-1">
		<div class="lab-step">First, calculate the total charge Q that flowed through the system during the different trials. One coulomb is the quantity of electricity flowing through a wire carrying 1 ampere of current in 1 s, thus Q = I x t.</div>
		</li>
		<li data-sub-note-item="2-2">
		<div class="lab-step">Review the current probe data for the first 5-min electroplating trial.<strong> Note: </strong>If the current dropped to 0 or close to 0 at the end of the trial, then the electrons stopped flowing from the copper electrode to the key. Only use the values recorded before the drop in current occurred. If the current drop to 0 early in the plating process, repeat the trial.</div>
		</li>
		<li data-sub-note-item="2-3">
		<div class="lab-step">The current varies over time, so calculate the average current, I.
		<h4><strong>Table 2: Moles of Electrons Transferred</strong></h4>
		<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
			<tbody>
				<tr>
					<td style="text-align: center;"><strong>Trial</strong></td>
					<td style="text-align: center;"><strong>Time (s) </strong></td>
					<td style="text-align: center;"><strong>Current<sub>average</sub> (A) </strong></td>
					<td style="text-align: center;"><strong>Total charge (C) </strong></td>
					<td style="text-align: center;"><strong>Moles (e?) </strong></td>
				</tr>
				<tr>
					<td style="text-align: center;">5 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">1<sup>st</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">2<sup>nd</sup> 30 min</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
				<tr>
					<td style="text-align: center;">Total</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
				</tr>
			</tbody>
		</table>
		<a href="https://www.jove.com/CDNSource/lm/labs/53/Table_2._Moles_of_Electrons_Transferred.xlsx" target="_blank">Click Here to download Table 2</a></div>
		</li>
		<li data-sub-note-item="2-4">
		<div class="lab-step">Use the average current over the total time to determine the total charge transferred during electroplating, Q. About 20 coulombs were transferred during the 5-min trial, and between 110 and 120 coulombs were transferred during each 30-min trial. The total charge transferred over the entire 65-min electroplating process was about 250 coulombs.</div>
		</li>
		<li data-sub-note-item="2-5">
		<div class="lab-step">Use the total charge transferred to calculate the moles of electrons plated during each trial. The moles of electrons plated equals the total charge transferred divided by the Faraday constant. The Faraday constant represents the magnitude of electric charge per mole of electrons and is determined by multiplying the charge of an electron by Avogadro&#39;s constant.</div>
		</li>
		<li data-sub-note-item="2-6">
		<div class="lab-step">Determine the moles of electrons that were transferred for the 5-min and 30-min trials.</div>
		</li>
		<li data-sub-note-item="2-7">
		<div class="lab-step">Calculate the moles of copper that were plated on the key after each trial. Similarly, determine the moles of copper lost from the copper sheet electrode.</div>
		</li>
		<li data-sub-note-item="2-8">
		<div class="lab-step">Compare the moles of copper that were lost from the electrode to the amount that was plated to the brass key. The numbers should be close.</div>
		</li>
		<li data-sub-note-item="2-9">
		<div class="lab-step">Check to see if these values match what is expected based on the number of moles of electrons that were transferred. The reduction of 1 mole of copper(2+) ions requires 2 moles of electrons to produce 1 mole of solid copper.</div>
		</li>
	</ul>
	</li>
</ol>

Electrolytic Cells: Construction and Electroplating - Procedure

Source: Smaa Koraym at Johns Hopkins University, MD, USA

	Electrolytic CellsExpand
	In this experiment, you will be assembling an electrolytic cell to ...

Extraction

Extraction: Liquid-Liquid and Acid-Base Extraction - Procedure

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

	Cellulose RecoveryExpand
	Extraction and filtration can ...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Human Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">In this experiment you will prepare different types of cells from a plant and animal, onion and human respectively, and then visualize them under a microscope. HYPOTHESES: The experimental hypothesis is that the cells will appear different in overall shape and membrane structure, but there will be some shared similar structures, like nuclei, in both cells. The null hypothesis of the experiment is that there will be no difference in structure between the two cell types.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">First, to prepare an animal cell slide, start by pouring 30 mL of distilled water into a beaker.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, use a glass dropper to place 2 - 3 drops of the water onto the center of a clean microscope slide.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Next, take a clean toothpick and scrape the inside of your cheek to gather cells.  CAUTION: Do not scrape your cheeks too aggressively. The scraping should not cause discomfort or bleeding. 
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Put the end of the toothpick with the cheek cells onto the wetted area of the slide and mix the water and cheek cells together.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Next, add two drops of methylene blue staining solution to the wetted area of the slide and mix it in with a clean toothpick.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Let the mixture sit at room temperature for 3 minutes.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After 3 minutes have passed, use a glass dropper to add a drop of glycerol to the mixture.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Carefully, place a cover slip over the mix on the slide by first standing one end of the cover slip vertically and aligning it along to one side of the mixture. Then, carefully lower the other end of the cover slip down until the mixture is completely covered.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Finally, remove any excess liquid with blotting paper.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Next, prepare a slide of plant cells by placing five drops of distilled water into a clean watch glass.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, with forceps, take a thin strip of onion and place it into the water.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Apply 5 drops of safranin solution to another watch glass.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Now using a pair of forceps transfer the piece of onion from the distilled water into the safranin.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Allow the onion to soak for 30 seconds.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, again with forceps, move the onion back into the distilled water.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Now, place 3 drops of glycerol onto the center of a glass microscope slide.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Using the forceps again, transfer the onion into the glycerol on the slide.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Gently place a cover slip over the onion on the slide and blot away any excess liquid with a piece of blotting paper.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">Next, you will visualize the slides you have prepared using a compound microscope. First, turn on the scope and set the objective lens to the lowest level of magnification.  NOTE: A compound microscope typically has two levels of magnification.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Place the cheek tissue slide onto the stage of the microscope and bring the cells into focus by adjusting the stage and using the coarse adjustment knob.
</div></li><li data-sub-note-item="1-22"><div class="lab-step">When the cells are in focus, observe the individual cells and draw what you see. Repeat these observations at medium and high magnifications adjusting the focus with the fine adjustment knob. IMPORTANT: Do NOT use the coarse adjustment knob at high magnifications. 
</div></li><li data-sub-note-item="1-23"><div class="lab-step">Next, rotate the nose piece so that the oil lens is almost in place.
</div></li><li data-sub-note-item="1-24"><div class="lab-step">Place a drop of immersion oil on top of the cover slip of the cheek cell slide.
</div></li><li data-sub-note-item="1-25"><div class="lab-step">Use the oil lens to view the cheek cells and record your observations.
</div></li><li data-sub-note-item="1-26"><div class="lab-step">Repeat these steps to view the onion plant cell, moving again from low to medium, high, and oil lenses (steps 20 &#8211; 24).
</div></li><li data-sub-note-item="1-27"><div class="lab-step">Record all of your observations and note the differences you see between animal and plant cells.
</div></li><li data-sub-note-item="1-28"><div class="lab-step">When you are finished, place the cheek cell slide and toothpicks into a diluted bleach solution. Dispose of the cover slips into a glass waste container.
</div></li><li data-sub-note-item="1-29"><div class="lab-step">Finally, turn off the microscope and clean the lenses with lens cleaning paper.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Calculate the total magnification for each of the viewing levels. Remember to account for both the eyepiece magnification and the objective lens magnification. NOTE: Eye piece: 10X magnification, Objective lens: 4X, 10X, and 40X magnification
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Then, identify the structural differences between plant and animal cells using the observations made under the microscope.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Label the nuclei in both cell types and then highlight or note any differences that you see between them.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Procedure

Visualizing Onion and Human Cells
ExpandIn this experiment you will prepare different types of cells from a plant and animal, onion and human ...

Biology

Fundamentals

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Observing the Cell Cycle in a Root Tip
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Hypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a chemical that interferes with microtubular polymerization, all of the cells will be arrested at the same stage of the cell cycle and that in untreated onion tip slices all of the different stages of the cell cycle will be visualized. The null hypothesis is that there will be no differences in mitosis between the two groups and that different stages of the cell cycle will be visualized. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">To prepare for this experiment you will use an onion that has been placed in water for several days until new white root tips have begun to sprout. Use a razor blade to cut a 1 cm piece from the end of one of the root tips. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the slice of onion into a microcentrifuge tube labeled &#8220;N&#8221; for the nocodazole condition. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, cut a second 1 cm piece of onion root tip and place in a microcentrifuge tube labeled &#8220;C&#8221; for the control condition.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Add 2 &#181;L of the 5 mg/mL nocodazole to 1 mL of water in the microcentrifuge tube labeled N and 1 mL of water only to the tube labeled C. 
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Leave the tubes containing the root tips to incubate for 12 hours and wipe down the workspace with 70% ethanol.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">When incubation is complete, set a heat block to 60 &#186;C.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Wash the nocodazole treated tips with water and then aspirate the liquid.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Repeat this wash two more times, for a total of three washes. 
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Fill both tubes with 1 mL of one normal hydrochloric acid. Incubate the tubes in a 60 &#186;C heat block for 12 - 15 min, then discard the acid into a waste flask.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Now, add 1 mL of distilled water drop wise to the tubes at least three times to rinse the samples.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Now add one microliter of DAPI stain to 10 mL of phosphate buffered saline (PBS).
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Then place 50 &#181;L of this dilute DAPI stain into each tube, and leave them at room temperature to incubate for 12 min. 
</div></li><li data-sub-note-item="1-14"><div class="lab-step">After this, preform three distilled water washes as in step 10. 
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Label one microscope slide as 'Control' and another as 'Nocodazole'. 
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Transfer the stained roots onto their respective microscope slides and add a drop of water to the control slide and a drop of nocodazole to the nocodazole slide.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Use the razor blade to remove the unstained portions of each root tip and place a glass cover slip over each sample.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Carefully press down on each cover slip with a gloved fingertip, and then let the slides sit for 10 min. 
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Finally, view the slides under the 40X objective of a fluorescence microscope and capture images of your root tip cell preparations.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Understanding the Loss of Cell Cycle Control
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">NOTE: Before cells can pass through all of the stages of the cell cycle there are several checkpoints they must go through. These checkpoints are regulated by cyclin and cyclin-dependent kinase proteins, or CDKs. If the cyclins and CDKs determine that previous cellular events have not been adequately completed they will arrest the cells at this step and prevent them from proliferating. In cancer cells one or several of these molecular restraints for regulating cell division are lost and this allows the damaged cells to escape from cell proliferation control. These abnormal cells develop into tumors which are masses of uncontrolled proliferating cells that interfere with the normal functions of the body. Notice how before it divides this cancer cell rounds up and refracts light very strongly under the light microscope. This is because not all cancer cells successfully divide into daughter cells after each round of mitosis. This will mean that they often contain an excess of organelles and DNA and this increase in cellular content means they will refract light more intensely than normal cells do. 
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Examine your collected images and record in Table 1 the different cell structures and stages that could be identified in the onion tip slice that was treated with nocodazole. <a href="https://www.jove.com/CDNSource/lm/labs/10/Table_1_Cell_division.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="3-2"><div class="lab-step">If there are any cells undergoing mitosis, note what stages of cell division you see.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Record the percentage of cells in each stage of mitosis in Table 1. 
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Next, examine the images of the untreated onion tip slice, and record what cell structures can you identify. 
</div></li><li data-sub-note-item="3-5"><div class="lab-step">If there are any cells undergoing mitosis, consider what stages of cell division you see, and record the percentages of cells in each stage of mitosis in the Table 1. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Procedure

Observing the Cell Cycle in a Root Tip
ExpandHypotheses: The experimental hypothesis is that in root tips slices that have been treated with nocodazole, a ...

Cellular Processes

Microbial and Fungal Diversity

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Examining Microbial Colony Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">To begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place the uncovered plate under a dissecting microscope and then observe the individual communities within each quadrant of the plate.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">As the colonies are identified, fill out the observed colony properties in the appropriate column in the table.  <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_1_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, repeat the observations for each of the other two bacterial species.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">To perform Gram staining of your three bacterial species, first label the ends of three microscope slides with the initials of each bacteria species, your initials, and the date. HYPOTHESES: In this experiment, the experimental hypothesis might be that it is possible to elucidate different bacterial species and shapes using Gram staining, and that Gram-positive bacteria will stain purple with crystal violet whereas Gram-negative bacteria will appear pink due to the safranin counterstain. The null hypothesis could be that all of the bacterial types will retain both stains equally, and staining will not help elucidate their species or shape.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">To begin the first stain, attach a clip to the slide for ease of handling. For each bacterial smear, remember to be sure to use a cleaned and flame-sterilized inoculating loop.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Place two loopfuls of sterile distilled water onto the center of the slide.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">After sterilizing the loop again, cool the loop by touching an uninoculated portion of the agar several times.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">Then, remove a small amount of culture from an isolated colony and mix it with the water on the slide.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Allow the slide to dry at room temperature and heat-fix the smear by quickly passing it through a flame.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">Once dry, pipette several drops of crystal violet onto the slide and let it sit for two to three minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, cover the slide with Gram&#39;s iodine, leave it to sit again for two minutes, and then gently rinse with distilled water.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Decolorize the slide with 95% ethanol until stain no longer washes away.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">Immediately, rinse the slide with distilled water. This will limit over-decolorizing.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add several drops of safranin counterstain and leave for 30 s. This will stain any Gram-negative cells present.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">Gently rinse the slide with distilled water and blot dry with absorbent paper.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">After staining the other two slides in the same manner (steps 6 &#8211; 17), observe each with a microscope.
</div></li><li data-sub-note-item="1-19"><div class="lab-step">Use a low power objective first to make coarse adjustments. When you find an ideal section, move on to the higher magnifications.
</div></li><li data-sub-note-item="1-20"><div class="lab-step">After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. NOTE: The oil is needed for high power objectives that will provide the best micrographs.
</div></li><li data-sub-note-item="1-21"><div class="lab-step">Now, use colored pencils to draw the observed bacteria in the appropriate magnification circles and fill in the observed characteristics in the appropriate column, noting the bacteria&#39;s shape and whether it is Gram-positive, identified by darker purple staining, or Gram-negative, identified with pink staining. <a href="https://www.jove.com/CDNSource/lm/labs/28/Table_2_Microbial_diversity.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="1-22"><div class="lab-step">Finally, repeat the observation, drawing, and characterization for the other two stained bacterial slides.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Examining Fungal Structures
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Remove the <em>S. cerevisiae</em> plate from the incubator and place a drop of water onto a microscope slide.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">Using a sterilized inoculating loop, smear a film of yeast into the drop of water and place a cover slip over the slide.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Place the slide under the microscope at the lowest magnification, increasing the magnification to the oil immersion setting to see the individual yeast cells.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Observe the general shape and structure of the yeast and identify any cells that are undergoing asexual reproduction by budding.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, draw the cells in the appropriate magnification circles.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">When you are done observing the yeast, replace the <em>S. cerevisiae</em> slide with a prepared Ascomycetes apothecium slide and set the microscope to the lowest magnification.
</div></li><li data-sub-note-item="2-7"><div class="lab-step">Using colored pencils, draw the structures at low and high magnifications, including the asci and ascospores.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">Finally, place a button mushroom, <em>Agaricus bisporus</em>, under the dissecting microscope and draw the features, including the pileus, or cap, the gills, and the stipe, or stalk.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">Using the coloring of the bacteria recorded in the table, determine which species are Gram-positive and which are Gram-negative.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Then, look at the shapes recorded in the table to determine which bacterial species are bacillus, coccus, or spirillum.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">Next compare your illustrations of the yeast cells to your illustrations of the bacterial cells. NOTE: Yeast from the Ascomycetes phylum, such as <em>S. cerevisiae</em>, can reproduce asexually, which can be observed through the budding of the cells under a microscope.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Record any observations of yeast cells budding.  NOTE: Ascomycetes can also reproduce sexually via mitosis and haploid spore formation.
</div></li><li data-sub-note-item="3-5"><div class="lab-step">Record any observations of spore or asci formation you observed in the table.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">NOTE: Fungus from the Basidiomycota phylum exhibit clearly observable gills under the pileus.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Record any fungal species that demonstrated gills under the pileus.
</div></li></ul></li></ol>

Microbial Colony and Fungal Diversity - Procedure

Examining Microbial Colony Structures
ExpandTo begin, collect your allocated culture plates and then remove the Parafilm and lid from one of them.
Place ...

Evolution

Macromolecules

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Identifying Macromolecules with Indicator Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">HYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an indicator dye to each test solution will result in a color change if the test solution contains the macromolecule of interest. The null hypothesis might be that the addition of an indicator dye will not result in a color change in any of the test solutions.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Place 16 clean test tubes in a tube rack in four rows of four tubes and label each row A through D and each column 1 - 4.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Label each test tube with its corresponding position in the test tube rack.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">After this, stir solution A thoroughly and use a pipette to add 4 mL of solution A to each tube in Row A.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Next, stir solution B until it is well mixed and use a new pipette to add 4 mL of solution B to each tube in Row B.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">After stirring solution C thoroughly, add 4 mL of solution C to each tube in Row C.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Then add 4 mL of soybean-derived vegetable oil to each tube in Row D.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">When all of the tubes have been filled with their respective test solutions, add 200 mL of water to a 400 mL beaker on a hotplate and set the hotplate to high.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">To perform the Benedict&#39;s Test, add 2 mL of Benedict&#39;s reagent to test tubes A1, B1, C1, and D1 while the water is warming.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Observe the initial color of the tubes.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">When the water begins to boil, transfer each test tube to the beaker and heat the tubes in the boiling water bath for 3 - 5 minutes.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Then, record the final color of each of the tubes in the appropriate cells of the table and turn off the hotplate. <a href="https://www.jove.com/CDNSource/lm/labs/22/Table_1_Macromolecules.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-13"><div class="lab-step">To perform an Iodine-Potassium-Iodide Test, first observe the color of the solutions in the test tubes in column 2.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Then, add 1 mL of iodine potassium iodide reagent to tubes A2 to D2 and record the final color change in each of the tubes.
</div></li><li data-sub-note-item="1-15"><div class="lab-step">For the Sudan IV Test, observe and record the color of the solutions in the test tubes in column 3.
</div></li><li data-sub-note-item="1-16"><div class="lab-step">Then, add 2 - 3 drops of Sudan IV reagent to test tubes A3 through D3 and record the color change, if any, in each tube.
</div></li><li data-sub-note-item="1-17"><div class="lab-step">For the Biuret Test, observe the color of the solutions in the test tubes in column 4.
</div></li><li data-sub-note-item="1-18"><div class="lab-step">Then, add 1 mL of Biuret reagent to test tubes A4 through D4 and record the final color in each of the tubes.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">Using the results from the indicator tests, each of the four original solutions can be identified as a sugar, starch, lipid, or protein. Refer to the Concepts video to help you determine which tubes contain which macromolecules.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">First, look at the results from the Benedict&#39;s Test. Determine which of the test solutions contain sugar based on your results.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">Next, create a bar graph with your five plant species listed on the x-axis and the surface area in cm indicated on the y-axis.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Next, look at the results from the Iodine-Potassium-Iodide Test. Determine which of the test solutions contain starch.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Now, review the results from the Sudan IV Test. Using these observations, determine if any of the test solutions contain lipids.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Finally, look at the results from the Biuret Test. Determine if any of the test solutions contain proteins.
</div></li></ul></li></ol>

Macromolecules: Identification Using Indicator Solutions - Procedure

Identifying Macromolecules with Indicator Solutions
ExpandHYPOTHESES: In this activity, the experimental hypothesis might be that the addition of an ...