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

Institutos nacionales de salud otorgar R01 CA149976 (N.A.S) apoyado este trabajo.

Todos los procedimientos deberán realizarse con arreglo institucional uso de animales y su cuidado. El procedimiento fue desarrollado para el análisis de murino madre y progenitoras células hematopoyéticas (HSCs y HPs), pero puede ser adaptado para el análisis de otras poblaciones de la célula.
1. flujo Cytometry aislamiento de HSCs murinas y HPs
<ol><li>Cosecha de células de médula ósea murina como se describe en la literatura6. Nota: En los datos present...

<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> J Immunol. 175 (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> Stem Cells. 30 (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> Nature. 509 (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> Cell. 148 (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> J Clin Invest. 122 (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> Genes Dev. 30 (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> Cell Stem Cell. , (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> Cell. 151 (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> Basic Methods in Cellular and Molecular Biology. , (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> Basic Methods in Cellular and Molecular Biology. , (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> Basic Methods in Cellular and Molecular Biology. , (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> Nat Rev Mol Cell Biol. 11 (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> J Vis Exp. (93), e52099 (2014).</li></ol>

Western Blot es una técnica común para la detección de proteínas específicas y la activación de vías de señalización en los tejidos o células. Mediante la introducción de pequeños ajustes a un procedimiento de uso general, hemos sido capaces de detectar rutinariamente 15 diversas proteínas (Tabla de materiales) en 15.000 HSCs y en algunos casos en tan solo 500 HSCs. The Most pasos críticos en este protocolo son: 1) exactamente contando las células 2) reduciendo al mínimo el número de tra...

Las células madre hematopoyéticas (HSCs) son células renueva a sí misma que pueden dar lugar a todos los linajes de sangre. Son células relativamente raras en la médula ósea, haciendo difícil los análisis bioquímicos. Enfoques adecuados para el análisis de células, como la citometría de flujo, han sido extremadamente útiles para cuantificar cantidades relativas de los marcadores de superficie celular y proteínas intracelulares. Sin embargo, el análisis de proteínas intracelulares requiere la utilización de procedimientos de permeabilización celular para permitir el acceso del anticuerpo, y no todos celular epitopos superficiales sobreviven estos procedimientos1,2. Además, anticuerpos que discriminan entre productos de diferentes proteínas isoformas o hendidura a menudo no están disponibles para citometría de flujo, y por lo tanto, los investigadores todavía dependen de Western blot para ciertos tipos de análisis.
Mancha blanca /negra occidental análisis de lysates de la célula es un procedimiento rutinario en la mayoría de los laboratorios. Las células se pueden purificar en condiciones nativas que conservan los epitopos de moléculas de superficie celular, y lysates de la célula puede preparado y analizado posteriormente. Sin embargo, el análisis de proteínas en célula de primaria rara poblaciones por Western blot puede requerir euthanizing grandes cantidades de animales para obtener suficientes células. Haciendo pequeños ajustes en varios pasos, un protocolo Blot Western convencional fue capaz de detectar proteínas en un número relativamente pequeño de HSCs (500-15.000, dependiendo de la proteína de interés). Los ajustes incluyen exactamente contando las células, manipular cuidadosamente el sedimento celular, reducir las transferencias de células entre tubos para minimizar la pérdida de la célula, y lisis de un número determinado de células con una carga concentrada tampón que contiene proteasoma y inhibidores de la fosfatasa. Muchos informes publicados incluyen manchas blancas /negras occidentales obtenidas con 20.000 o más HSCs3,4,5,6,7; Este sencillo procedimiento reducirá el número de células y animales de experimentación necesarios para producir datos equivalentes por entre 4 y 40 veces. El protocolo está diseñado para normalizar los resultados en una base por células, en lugar de un control interno. Esto permite la detección de la reducción general en los niveles de proteína que pueden pasarse por alto si los datos se normalizan a un control interno. La importancia de la normalización de una base por celular fue descrita para el análisis de de datos de expresión génica8, y el mismo principio se aplica a la cuantificación de proteínas por Western blot. Este protocolo optimizado debe ser útil para cualquier persona que necesite analizar una pequeña cantidad de células.

<table>
	<tbody>
		<tr>
			<td>sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166-500</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Fisher Scientific</td>
			<td>BP150-20</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>30% Acrylamidel Bis solution</td>
			<td>Bio-Rad</td>
			<td>1610158</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>1.5M Tris-HCl pH8.8</td>
			<td>TEKNOVA</td>
			<td>T1588</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>1.0M Tris-HCl pH6.8</td>
			<td>TEKNOVA</td>
			<td>T1068</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>BP179-25</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>mini-protean 3 comb</td>
			<td>Bio-Rad</td>
			<td>1653360</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td> 
			Mini-PROTEAN Tetra Cell</td>
			<td>Bio-Rad</td>
			<td>1658005EDU&#160;</td>
			<td>&#160;SDS-PAGE</td>
		</tr>
		<tr>
			<td>&#160;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&#160;</td>
			<td>electrophoresis</td>
		</tr>
		<tr>
			<td>Imaging System</td>
			<td>Bio-Rad</td>
			<td>1708195&#160;</td>
			<td>signal detection</td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610747EDU&#160;</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>10x Tris/Glycine/SDS Electrophoresis Buffer</td>
			<td>Bio-Rad</td>
			<td>1610732EDU&#160;</td>
			<td>electrophoresis</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>1610710EDU</td>
			<td>sample preparation</td>
		</tr>
		<tr>
			<td>&#160;RTA Mini PVDF Transfer Kit, for 40 blots</td>
			<td>Bio-Rad</td>
			<td>1704272&#160;</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 
			antibody concentration: 1:1000 
			reference(figure): Fig2</td>
		</tr>
		<tr>
			<td>p-Rps6</td>
			<td>Cell signaling</td>
			<td>4858</td>
			<td>WB antibody,validated in 1000 cells 
			antibody concentration: 1:1000 
			reference(figure): Fig2</td>
		</tr>
		<tr>
			<td>actin(HRP conjugated)</td>
			<td>abcam</td>
			<td>ab49900</td>
			<td>WB antibody,validated in 500 cells 
			antibody concentration: 1:5000 
			reference(figure): Fig1, Fig2</td>
		</tr>
		<tr>
			<td>LC-3</td>
			<td>Abcam</td>
			<td>ab48394</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig5)</td>
		</tr>
		<tr>
			<td>S6</td>
			<td>Cell Signaling</td>
			<td>2317</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>4EBP1</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>WB antibody, validated in 15000 cells 
			antibody concentration: 1:1000 
			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 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>TSC2</td>
			<td>Cell Signaling</td>
			<td>4308</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig4)</td>
		</tr>
		<tr>
			<td>HSP90</td>
			<td>Cell Signaling</td>
			<td>4877</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (Fig5)</td>
		</tr>
		<tr>
			<td>PTEN</td>
			<td>Cell Signaling</td>
			<td>9188</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>mTOR</td>
			<td>Cell Signaling</td>
			<td>2983</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			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 
			antibody concentration: 1:1000 
			reference(figure): ref5 (FigS1)</td>
		</tr>
		<tr>
			<td>PP2AB</td>
			<td>Cell Signaling</td>
			<td>4953</td>
			<td>WB antibody,validated in 15000 cells 
			antibody concentration: 1:1000 
			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 
			antibody concentration: 1:1000 
			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 
			antibody concentration: 1:3000 
			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>&#160;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>&#160;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

Las células madre hematopoyéticas (HSCs) son las células raras, con la médula ósea de ratón con sólo 25.000 ~ fenotípica largo plazo HSCs repoblamiento. Un Western Blot protocolo fue optimizado y adecuado para el análisis de una pequeña cantidad de HSCs (células 500-15.000). HSCs fenotípicas fueron purificadas, contó con precisión y lisis directamente en Tampón Laemmli. Lisados que contiene igual número de células se analizaron por electroforesis en gel de poliacrilamida sodio dodecil sulfato (SDS-PAGE), y lo blot fue preparado y procesadas siguiendo el estándar de Western Blot protocolos. Mediante este protocolo, 2.000-5.000 HSCs pueden analizarse de forma rutinaria, y en algunos casos pueden obtenerse datos de tan sólo 500 células, en comparación con las células de 20.000 a 40.000 registradas en la mayoría de las publicaciones. Este protocolo debe aplicarse generalmente a otras células hematopoyéticas y permite el análisis de rutina de un pequeño número de células utilizando procedimientos estándar del laboratorio.

Resultados representativos de 500-2.000 purificada HSCs y HPs se muestran en la figura 1 y figura 2. La señal de β-actina en la figura 1 se puede detectar desde tan sólo 500 HSCs y HPs purificaron de la médula ósea de un ratón. Tenga en cuenta que cargar los lisados en pozos de 1.5 mm produce una señal mucho más fuerte de 500 HPs que carga en pocillos de 3,0 mm. Figura 2<...

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

Un estándar de Western Blot protocolo fue optimizado para el análisis de tan sólo 500 madre hematopoyética o células progenitoras. Optimización implica la cuidadosa manipulación de la muestra de células, limitando las transferencias entre los tubos y directamente lisis de las células en Tampón Laemmli.

Un Western Blot protocolo para una pequeña cantidad de células madre hematopoyéticas

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

The over goal of this western blotting protocol is to Perform western blots on small numbers of cells. This method can help answer key questions in the hematopoietic stem cell field such as the presence or abundance of specific proteins or activation of signaling pathways. The main advantage of this technique is that it requires fewer than 20, 000 cells thus fewer animals are needed to analyze rare populations of primary cells.We first had the idea for this method when we were trying to find ways to reduce the number of mice we needed to use for hematopoietic stem cell analyses. Demonstrating the procedure will be Xiongwei Cai, a post doc from my laboratory. Use a swinging bucket rotor to centrifuge 1.5 milliliter tubes containing sorted hematopoietic stem and progenitor cells at 500 times gravity for five minutes at four degrees Celsius.Once the centrifugation is over, discard all but 100 microliters of supernatant, without disturbing the cell pellet. If there are less than 5, 000 cells, and the volume of the sorted sample is less than 0.2 milliliters, first transfer the cells to low binding 0.2 milliliter PCR tubes before centrifugation. However, if there are less than 5, 000 cells, but the volume of the sorted sample is more than 0.2 milliliter, then centrifuge the cells at 500 times gravity for five minutes at four degrees Celsius.After centrifugation, discard all but 200 microliters of supernatant. Then re suspend the pellet and the remaining 200 microliters of supernatant. Next, transfer the cell suspension to 0.2 milliliter PCR tubes and again, centrifuge.Then re suspend the pellet in 20 to 30 microliters of supernatant and discard the rest. If necessary, add phosphate buffered saline in 2%fetal bovine serum, or 2%bovine serum albumin to the re suspended cells. Then use an automated cell counter to determine the cellular concentration using two to five microliters of re suspended cells.Next use a 20 or 100 microliter pipette tip to transfer a volume of 500, 1, 000, and 2, 000 hematopoietic stem and progenitor cells to new 1.5 milliliter tubes. Use a swinging bucket rotor to centrifuge the cells at four degrees Celsius at 500 times gravity for five minutes. Then follow the manufacturers instructions to dissolve phosphatase and proteasome inhibitor and DMSO to prepare a 100 fold stock solution.Next dilute the four fold Laemmli sample buffer with equivalent volumes of distilled water to make a two fold Laemmli sample buffer. Then add appropriate amounts of the 100 fold stock solution of phosphatase and proteasome inhibitor to adjust the final concentration to two fold inhibitors, and two fold Laemmli sample buffer. Adjust the volume of residual supernatant in the tube such that adding equal volumes of two fold Laemmli buffer along with the phosphatase and proteasome inhibitor will achieve a final concentration of 500 cells per microliter and one fold Laemmli buffer.And then re suspend to form the lysate. Before loading the gel, heat the sample lysates at 95 degrees Celsius in a heat block for five minutes. Using standard protocols electrophoresis one to 40 microliters of lysate equivalent to 500 to 20, 000 cells through SDS page at 100 volts.After treating poly vinylidene membrane with methanol for then seconds wash the membrane with distilled water briefly. Then transfer the protein from the gel to the membrane according to the instructions provided by the manufacturer of the transfer apparatus. After transfer, block the membrane with two milliliters of 2%bovine serum albumin and phosphate buffered saline with 0.1%Tween overnight at four degrees Celsius according to standard protocals.After blocking the membrane, incubate it with diluted antibodies overnight at four degrees celsius. Then wash the membrane thrice with phosphate buffered saline with 0.1%Tween 20 for five minutes each at room temperature. Next incubate the membrane with two milliliters of enhanced chemiluminescence buffer for one minute at room temperature.Detect the signals using an imaging system. To compare the beta actin signals lysates were prepared from urine hematopoietic stem and progenitor cells and subjected to western plot analysis. It is clear form the amino blot that there is a decrease in beta actin signal from the lysates prepared from 2, 000 to 500 hematopoietic progenitor and stem cells loaded in 1.5 millimeter wells on a 12%SDS page gel.Further, to compare the beta actin signals obtained from different well sizes cast on the gel. Lysates were loaded in 1.5 and three millimeter wells on the gel and then subjected to western blot analysis. The blot shows that the beta actin signal obtained from the lysate prepared from 500 hematopoietic progenitors loaded in a 1.5 millimeter well is much stronger than when loaded in a three millimeter well.Next, to compare the signals for eucaryotic initiation factor 4G, actin and phosphorylation of ribosomal protein S6 in response to cytokine stem cell factor stimulation the lysates obtained were analyzed by amino blot. The blot shows the presence of stronger signal intensities for all three proteins in the hematopoietic re genitors with cytokine stem cell factor stimulation. In comparison to the control in vitro it is also clear that there is and increasing signal intensity for all three proteins from different hematopoietic stem cell volumes.Once mastered this technically can be done in 48 hours. If it is approached properly. While attempting this procedure it's important to remember to accurately count the cells and to direct the lysate cells with a loading buffer.After this development it is technically pelleted away for the researchers in the field of stem cell research to explore signal pathways in small number of cells. After watching this video you should have a good understanding of how to do western plotting with a small number of address pieces. Be careful with the regions you're using for SDS page.Thank you for watching. Good luck with your experiments.

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

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Investigación

Bioquímica

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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

Una adaptación de semi-alto rendimiento del ensayo ATPase acoplado a NADH para la detección de inhibidores de moléculas pequeñas

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

A Rapidly Incremented Tethered-Swimming Maximal Protocol for Cardiorespiratory Assessment of Swimmers

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A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Un ensayo de actividad acoplada a enzimas de alto rendimiento para sondear la interacción de moléculas pequeñas con la dNTPasa SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Desarrollo de una tira inmunocromatográfica de flujo lateral para la detección rápida y cuantitativa de compuestos de moléculas pequeñas

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

Generación de organoides del corazón humano autoensamblables derivados de células madre pluripotentes

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Síntesis de masarimicina, un inhibidor de molécula pequeña del crecimiento bacteriano grampositivo

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...