We would like to thank all the members of the McMahon laboratory for their assistance and support.&#160;This research was supported by grants from an NIH/NCI R01 CA176839-01 (to MM) and an Institutional Research and Academic Career Development Award (IRACDA to JS).&#160;

1. Preparation of Whole Cell Lysates from Cell Culture
<ol>
	<li>Place cell culture dishes on ice and add cold PBS with 5 mM EDTA (e.g. 5 ml of PBS with 5 mM EDTA/10 cm2 plate). Remove adherent cells from the dish using a cell scraper and then transfer the cell suspension to a cold 15 ml conical tube.</li>
	<li>Pellet the cell suspensions by low-speed centrifugation at 1,000 rpm (230 x g) for 5 min at 4 &#176;C. Place conical tubes on ice and carefully aspirate the supernatant without disrupting the pellet.</li>
	<li>Wash the pellet with 1 ml of PBS with 5 mM EDTA and transfer cell suspension to a cold 1.5 ml centrifuge tube. Quick spin suspension at 13,000 rpm (13,226 x g) for 10 sec to pellet cells. Carefully remove supernatant without disrupting the pellet and place tubes on ice.</li>
	<li>Resuspend the pellet in 25-200 &#956;l (depending on the pellet size; i.e. for 3 x 106 cells add ~150 &#956;l of RIPA buffer ) in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) containing freshly added protease and phosphatase inhibitors at a 2x final concentration. Briefly vortex each tube and incubate suspension&#160;for 20-30 min on ice. Note: there are several lysis buffers that can be used to extract proteins, which differ in their ability to solubilize proteins and strength of their denaturing detergent (i.e. SDS, Triton X-100, or NP-40). RIPA buffer is useful for preparing whole cell lysates and disrupting protein-protein interactions.</li>
	<li>To create a post-nuclear fraction, centrifuge lysates at 14,000 rpm (15,339 x g) for 5 min&#160;at 4 &#176;C. Transfer the supernatant to a new tube without disrupting the nuclear-enriched pellet and place tubes on ice. Note: the nuclear-enriched pellet may also detach from the tube while extracting the supernatant. To avoid collecting the nuclear-enriched pellet, place the pipette tip directly into the viscous nuclear-enriched pellet and draw it up with the pipette in order to dispose of it.</li>
	<li>Determine the protein concentrations of each sample according to the manufacturer&#39;s instructions using such protein quantification assays as BCA or Bradford.</li>
</ol>
2. Sample Preparation and Electrophoresis
<ol>
	<li>Prepare each sample according to the following chart outlined below. As an alternative, &#946;-mercaptoethanol can also be used at a final concentration of 2.5%. However, the reducing agent should be added to each sample up to an hour before loading the gel. The total volume accounts for pipetting variations with only 20 &#956;l of each prepared sample loaded per well.
	<table border="1">
		<tbody>
			<tr>
				<td>REAGENT</td>
				<td>REDUCED SAMPLE</td>
			</tr>
			<tr>
				<td>Protein Sample</td>
				<td>X &#956;l</td>
			</tr>
			<tr>
				<td>4x LDS Sample Buffer</td>
				<td>6 &#956;l</td>
			</tr>
			<tr>
				<td>500 nM DTT Reducing Agent</td>
				<td>2.4 &#956;l</td>
			</tr>
			<tr>
				<td>Deionized Water</td>
				<td>Up to 15.6 &#956;l</td>
			</tr>
			<tr>
				<td>Total Volume</td>
				<td>24 &#956;l</td>
			</tr>
		</tbody>
	</table>
	</li>
	<li>Heat samples at 70 &#176;C for 10 min. While the samples are heating, prepare 1,000 ml of 1x MES or MOPS Running Buffer (50 ml 20x MES or MOPS Running Buffer plus 950 ml deionized water). MOPS Running buffer resolves mid-size proteins between 14-200 kDa, whereas MES Running Buffer resolves smaller molecular weight proteins between 2-200 kDa with a lower pKa, which allows the MES Running Buffer to run faster than the MOPS4. Thus, these two buffers have contrasting effects on the migration of ions within the stacking gel that leads to differences in the separation of the protein bands4. Set aside and combine 200 ml of 1x MES or MOPS Running Buffer with 500 &#956;l of Antioxidant, which will be used to fill the Upper Buffer Chamber of the Mini-Cell electrophoresis unit.</li>
	<li>Once the samples are finished heating, quick spin samples before placing them on ice.</li>
	<li>For assembly of the precast Bis-Tris gels into the Mini-Cell unit for electrophoresis follow the manufacturer&#39;s instructions. Note: make sure each gel is oriented in such a fashion that the shorter (smaller) cassette of each gel faces inward toward the Buffer Core and the gels are level and tightly pressed against the Buffer Core to avoid leakage from the Upper Buffer Chamber.</li>
	<li>Fill the Upper Buffer Chamber with the 200 ml of 1x MES or MOPS Running Buffer containing the Antioxidant and the Lower Buffer Chamber of the Mini-Cell unit with approximately 600 ml of 1x MES or MOPS Running Buffer. The extra 200-300 ml of running buffer can be saved for later use. However, it is not recommended to recycle the buffer used during the electrophoresis run as the pH and quality of the buffer may be altered.</li>
	<li>Load 20 &#956;l of each protein sample into each well. If sectioning the membrane into various strips in order to probe for multiple antibodies (i.e. more than 2), it is highly recommended to load 2-5 &#956;l of the prestained protein standard in the first and last wells of each gel as this will serve as cutting guides for separating the different sections of the membrane without affecting the imaging of the protein bands.</li>
	<li>Run gels at 200 V constant for 35 min with an expected current of 110-125 mA/gel at the start and 70-80 mA per gel at the end. Electrophoresis is complete when the tracking dye migrates to the platinum wire along the Buffer Core.</li>
</ol>
3. Protein Transfer Using the iBlot Dry Blotting System
<ol>
	<li>Completely separate the mini-gels by breaking the bonded sides of the cassette (this may produce a crackling noise). Discard the shorter (smaller) plate and carefully transfer the gel from the longer (slotted) plate into a container with deionized water and remove the thick-footed portion of the gel located at the bottom.&#160;Note: on rare instances, the gel may attach to the shorter plate instead of the longer, slotted plate.&#160;In this case, discard the longer, slotted plate and remove the thick-footed portion of the gel located at the bottom.&#160;Then, carefully transfer the gel from the shorter plate to deionized water.</li>
	<li>Assemble the anode and cathode transfer stacks according to the manufacturer&#39;s instructions. Note: both nitrocellulose or PVDF membranes provide high-quality and efficient transfer of proteins and are compatible with fluorescent detection8. However, PVDF membrane does have a higher binding capacity (240 &#956;g/cm3) than nitrocellulose (200 &#956;g/cm3)8.</li>
	<li>Carefully remove any bubbles or air trapped between the gel and membrane as this will prevent any distortion of the protein bands during the transfer. Note: do not trim the membrane or the transfer stack to fit your gel size as this will cause direct contact between the anode and cathode stacks.</li>
	<li>After properly assembling the transfer stacks on the dry blotting device, select the appropriate voltage program (i.e. Program 3: 20 V for 7 min) and begin the transfer. Note: proteins greater than 150 kDa tend migrate more slowly and may require a longer transfer time of 8-10 min. In addition, prior incubation of the gel in 20% ethanol for 5-10 min will also help to improve the transfer efficiency of these large proteins. Once the transfer program is complete, the device will automatically shut off the current. It is best to immediately remove the membrane and proceed with the blocking procedure to ensure better protein detection and avoid drying out the membrane.</li>
</ol>
4. Infrared Fluorescent Protein Detection
<ol>
	<li>Block membrane(s) in a minimum of 0.8 ml/cm2 of Odyssey Blocking Buffer (Do Not Add Tween-20) for 1 hr or overnight at 4 &#176;C. Membrane(s) can also be blocked with nonfat dry milk, however this may cause higher background and interfere with protein detection.</li>
	<li>Incubate membrane(s) in primary antibody at the desired concentration in Odyssey Blocking Buffer with 0.1% Tween-20 overnight at 4 &#176;C. For two-color detection, in which two different antigens are visualized on the same blot, the two primary antibodies must be derived from different host species and added simultaneously to the membrane(s).&#160;Note: prior to incubating the membrane(s) in primary antibody, the membrane(s) can be sectioned into various pieces in order to probe for multiple antibodies (i.e. more than 2) on the same membrane.</li>
	<li>Wash membrane(s) 3x for 10 min in TBS plus 0.1% Tween-20 with gentle shaking.</li>
	<li>Incubate membrane(s) with the fluorescently-labeled secondary antibodies at a dilution of 1:20,000 for the 700 nm channel and 1:6,000-1:10,000 for the 800 nm channel in Odyssey Blocking Buffer with 0.1% Tween-20 for 30-60 min (incubating membrane(s) for longer than 1 hr may increase background). Protect membrane(s) from light.&#160;Note: the secondary antibodies need to be derived from the same host species as each of the primary antibodies and labeled with different fluorophores.</li>
	<li>Wash membrane(s) 3x for 10 min in TBS plus 0.1% Tween-20 with gentle shaking. Protect membrane(s) from light.</li>
	<li>Membrane(s) may be dried or stored in either TBS or PBS without Tween-20 at 4 &#176;C until scanned and imaged with an Infrared Imaging System. Fluorescent signal will remain stable for several months if properly protected from the light.</li>
</ol>

<ol>
	<li>Burnette, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate--polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Anal. Biochem. 112, 195-203 (1981).</li><li>Renart, J., Reiser, J., Stark, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 3116-3120 (1979).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354 (1979).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NuPAGE Technical Guide. Life Technologies Corporation. IM-1001. 60, (2010).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Kubo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+incubation+of+solutions+of+proteins+containing+dodecyl+sulfate+on+the+cleavage+of+peptide+bonds+by+boiling.">Effect of incubation of solutions of proteins containing dodecyl sulfate on the cleavage of peptide bonds by boiling.</a> Anal. Biochem. 225, 351-353 (1995).</li><li>Moos, M., Nguyen, N. Y., Liu, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+high+yield+sequencing+of+proteins+electrophoretically+separated+and+transferred+to+an+inert+support.">Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support.</a> J. Biol. Chem. 263, 6005-6008 (1988).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iBlot+Dry+Blotting+System.">iBlot Dry Blotting System.</a> Life Technologies Corporation. 25-0911, 84 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blot+Analysis.">Western Blot Analysis.</a> LI-COR Biosciences. 988-12622, (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odyssey+Infrared+Imaging+System+Application+Software+Version+3.030.">Odyssey Infrared Imaging System Application Software Version 3.030.</a> LI-COR Biosciences. , (2007).</li><li>Hambaeus, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Milk+Composition.">Human Milk Composition.</a> Clin. Nutr. 54, 219-236 (1984).</li><li>DenHollander, N., Befus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+antigens+from+immunoblotting+membranes.">Loss of antigens from immunoblotting membranes.</a> J. Immunol. Methods. 122, 129-135 (1989).</li></ol>

The authors have nothing to disclose.

The critical steps in generating high-quality, quantitative Western blots according to this protocol are the following: 1) measuring protein concentrations; 2) sample preparation; 3) blocking the membrane and; 4) quality and caliber of the primary antibody.
The accuracy of measuring total protein concentrations can have a major impact on quantifying protein bands since the exceptional sensitivity of infrared fluorescent detection will amplify any slight differences found in the protein concentration of the samples. To avoid discrepancies in determining the protein concentration, generating a standard curve with a linear regression line or R-squared value as close to 1 as possible (R2&#8805;0.99 or better) is important in the precise calculation of the protein concentration of each sample. For instance, if the R2 value falls below 0.99, preparing fresh BSA standards with ultrapure water and carefully repeating the protein quantification assay may help to increase the R2 value. It is also best to avoid using a multichannel pipettor as this will generate inconsistencies in pipetting that may contribute to reduced R2 values and inaccuracies in the protein concentrations of the samples.
Sample preparation plays a significant role in determining the quality of the protein bands, which can also have a direct affect on the quantification of these bands. When using the Bis-Tris gel system, it is highly recommended that samples are prepared with LDS Sample Buffer because the slightly alkaline pH (8.5) of this buffer provides the optimal condition for the reduction of disulfide bonds and denaturation4. This sample buffer also contains a Coomassie G250 tracking dye that produces a sharp dye front that migrates with the moving ion front, which helps to ensure that small peptides do not run off the gel4. More importantly, the LDS Sample Buffer minimizes the cleavage of aspartyl-prolyl peptide bonds when samples are heated at 70 &#176;C due to the pH of the buffer changing from 8.5-8.04. This results in an ideal environment for the reduction and alkylation of proteins while also preserving the integrity of the protein. However, if a Tris-Glycine SDS Sample Buffer is used instead of the LDS Sample Buffer, this will lead to the cleavage of the aspartyl-prolyl bonds, a decrease in the sharpness of the protein bands, and an increase protein fragmentation4. It is also pertinent to add the Antioxidant to the Upper Buffer Chamber of the Mini-Cell electrophoresis unit. Since the Antioxidant is able to comigrate with the samples at neutral pH in the Bis-Tris gel system, this can help maintain proteins in a reduced state and protect disulfide bonds as well as sensitive amino acids against oxidation, whereas the absence of the Antioxidant can lead to diffused protein bands4. Lastly, it is highly recommended to use either MES or MOPS Running Buffer with Bis-Tris gels as opposed to using Tris-Glycine SDS Running Buffer with these types of gels. The combination of Tris-Glycine SDS Running Buffer and Bis-Tris gels results in the slow migration of glycine ions causing a dramatic increase in the electrophoresis run time with bands appearing more compressed and cup-shaped4.
The blocking step is also crucial for producing high-quality Western blot images using fluorescent detection as many of the various blocking agents can generate high membrane background. For infrared fluorescent detection, it is best to use the Odyssey Blocking Buffer because this specific blocking agent increases the sensitivity of protein detection while maintaining low background. Although nonfat dry milk, casein, or BSA could also be used as alternative blocking agents, these types of solutions can cause higher membrane background and in some cases a decrease in the binding affinity of the primary antibody10. In addition, milk-based blockers may contain IgG that can cross-react with phospho-epitopes and anti-goat antibodies, which could interfere with the accuracy of protein detection and proper antibody binding9,11. However, if a Western blot image is plagued by high background, nonspecific bands, or weak/no signal, then utilizing one of these alternative blocking agents may help to improve the quality of the image. Moreover, it is also recommended to avoid adding detergent to the blocking step and long blocking incubations as this may lead to an increase in background and/or loss of the target protein from the membrane9,12 .
Simultaneously detecting two different antigens on the same blot using infrared antibodies labeled with a 700 nm and 800 nm channel dye requires prudent selection of both the primary and secondary antibodies. The two primary antibodies must be derived from different host species. For instance, if one primary antibody is derived from rabbit, then the second primary antibody needs to be derived from a different species other than rabbit, such as mouse. Similarly, the infrared secondary antibodies also need to be derived from the same host species as each of the primary antibodies and labeled with different fluorophores. For example, a goat anti-rabbit in the 800 nm channel can be combined with a goat anti-mouse in the 700nm channel in order to accurately observe the protein signal from two different primary antibodies that are derived from rabbit and mouse.
Primary antibodies vary considerably in their quality, affinity, and sensitivity for their antigen. This can result in a significant reduction in signal or an increase in the nonspecific binding of proteins to the membrane thereby creating difficulties in accurately visualizing the target protein. Thus, by using an alternative blocking solution, such as nonfat dry milk or casein dissolved in PBS, may help to improve the protein detection with poor-quality primary antibodies10. In addition, the detergent concentration can also affect the binding of the primary antibody to the target antigen and precaution should be considered when determining the concentration of detergent in order to avoid washing away the antibody. A low concentration of SDS (0.01-0.02%) can reduce background and nonspecific binding when added to the diluted secondary antibody, especially when using PVDF membrane10. However, SDS can also disrupt antigen-antibody binding leading to a severe reduction in signal strength.
Furthermore, the novel modifications described in this protocol exemplifies a modern approach to the traditional Western blotting technique. These cutting-edge advancements drastically improve the efficacy, consistency, and sensitivity of Western data and allows for the accurate quantification of multiple target proteins on the same membrane regardless of their signal strength. More importantly, these valuable alterations also significantly reduce the experimental time of performing a Western blot while producing high qualitative and quantitative data.

The technique of Western blotting was first developed between 1977 and 1979 in order to create a better method for detecting proteins using antibodies1-3. This procedure utilized electrophoretic transfer of proteins to membranes from SDS-PAGE gels with target proteins visualized using secondary antibodies and detected by autoradiography, UV light, or a peroxidase reaction product3. Thus, these same basic principles are still widely used in today&#39;s Western blot protocols. However, this classic Western blotting technique does present many disadvantages, such as slow electrophoresis run times, low resolution and artificial protein bands, susceptibility to protein degradation, and limited sensitivity as well as poor data quality4. Therefore, this protocol describes significant advances and improvements to the standard Western blot procedure that generates more accurate qualitative and quantitative data.
The Laemmli system for separating a broad range of proteins using SDS-PAGE is the most widely used gel system for Western blotting5. Despite the popularity of this Western blotting system, this method can result in band distortion, loss of resolution, and spurious bands4. This may be a consequence of the deamination and alkylation of proteins due to the high pH (9.5) of the separating gel, reoxidation of reduced disulfide bonds due to the varying redox state of the gel, and cleavage of aspartyl-prolyl peptide bonds due to heating the protein in Laemmli buffer (pH 5.2)4,6. The Bis-Tris gel system, which operates at a neutral pH (7.0), provides significant benefits over the Lamemmli system. This system improves protein stability, minimizes protein modifications, maintains proteins in their reduced states, prevents aspartyl-prolyl cleavage during electrophoresis, and more importantly, the electrophoresis run time is 35 min4,6,7. In addition, the Bis-Tris gel system also produces sharper bands, higher resolution and separation, and increased sensitivity resulting in more reliable data4.
In conjunction with the Bis-Tris gel system, the iBlot dry blotting system uses high field strength and currents to significantly reduce the transfer time of proteins from gels onto membranes within 7 min8. This transfer system is based on the dry blotting method that generates a more efficient and reliable transfer of proteins8. For a detailed comparison of the efficacy of the iBlot transfer system to the conventional transfer systems please refer to the following website: <a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Western-Blotting/Western-Blot-Transfer/iBlot-Dry-Blotting-System/iBlot-Dry-Blotting-Comparison-to-Semi-Dry-and-Wet.html">http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Protein-Expression-and-Analysis/Western-Blotting/Western-Blot-Transfer/iBlot-Dry-Blotting-System/iBlot-Dry-Blotting-Comparison-to-Semi-Dry-and-Wet.html</a>. It utilizes an anode and cathode stack, which are comprised of a gel matrix that contains the appropriate transfer buffers, that act as ion reservoirs8. During the transfer, water electrolysis helps to prevent the generation of oxygen from the copper anode resulting in a more consistent protein transfer without causing band distortion8. Moreover, this transfer system also increases the transfer speed by reducing the distance between the electrodes8.
Although chemiluminescence is the most common and traditional technique for protein detection of Western blot analyses, two-color infrared fluorescent detection greatly improves the sensitivity, quality, and accuracy of Western blot data. This detection method uses infrared laser excitation in two optimal wavelengths, 700 nm and 800 nm, to generate a clear data image with the greatest signal-to-noise ratio and highest sensitivity9. Thus, two target proteins can be visualized simultaneously on the same membrane using the 700 nm and 800 nm fluorescent detection channels. More importantly, the linearity and dynamic range of infrared fluorescence allows for precise quantitative analysis of both strong and weak protein bands9. Furthermore, this protocol provides tremendous advantages and improvements over the classic Western blotting technique by decreasing the electrophoresis and transfer times of this experiment without compromising the efficacy of these processes and utilizing infrared fluorescent protein detection to produce greater qualitative and quantitative Western blot data.

<table border="0" cellpadding="0" cellspacing="0" width="624">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:297px;">BCA Protein Assay Kit</td>
			<td style="width:204px;">Thermo Scientific/Pierce</td>
			<td style="width:123px;">23225</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">4x NuPAGE&#174; LDS Sample Buffer&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0007</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">10x NuPAGE&#174; Sample Reducing Agent&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0004</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20x NuPAGE&#174; MES SDS Running Buffer</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0002</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20x NuPAGE&#174; MOPS SDS Running Buffer</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0001</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">NuPAGE&#174; Antioxidant</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0005</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:297px;">NuPAGE&#174; Novex&#174; 4-12% Bis-Tris Gel 1.5 mm, 10 well&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0335BOX</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:297px;">NuPAGE&#174; Novex&#174; 4-12% Bis-Tris Gel 1.5 mm, 15 well</td>
			<td>Invitrogen/Life Technologies</td>
			<td>NP0336BOX</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">SeeBlue&#174; Plus2 Pre-Stained Standard&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>LC5925</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">XCell SureLock&#174; Mini-Cell&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>EI0001</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Transfer Stack, PVDF Regular</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB4010-01</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174;Transfer Stack, PVDF Mini</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB4010-02</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Transfer Stack, Nitrocellulose Regular</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB3010-01</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174;Transfer Stack, Nitrocellulose Mini</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB3010-02</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iBlot&#174; Gel Transfer Device&#160;</td>
			<td>Invitrogen/Life Technologies</td>
			<td>IB1001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-Actin</td>
			<td>Sigma</td>
			<td>A2228</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phospho-BIM (S69)</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Total BIM</td>
			<td>Epitomics</td>
			<td>1036-1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phospho-ERK1/2 (T202/Y204)</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Total ERK1/2</td>
			<td>Cell Signaling Technology</td>
			<td>9107</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Blocking Buffer&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>927-40000&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">IRDye 800CW Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-32211&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">IRDye 680LT Goat anti-Mouse IgG&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>926-68020&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Western Incubation Box, Medium&#160;</td>
			<td>LI-COR Biosciences</td>
			<td>929-97205&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Classic Imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>N/A</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Odyssey&#174; Application Software V3.0.30</td>
			<td>LI-COR Biosciences</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>

the fastest western in town: a contemporary twist on the classic western blot analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

Two-color infrared fluorescence detects both strong and weak bands on the same membrane with enhanced sensitivity and the highest signal-to-noise ratio to produce clear Western blot images. Typical results generated by two-color Western blot detection visualized in both the 700 nm and 800 nm fluorescent channels are exemplified in Figures 1 and 2. The uppermost Western blot in Figure 1 shows the concurrent (overlay) detection of total ERK1/2 in the 800 nm channel (green) and &#946;-Actin in the 700 nm channel (red) of two-fold serial dilutions of lysates from the human-derived melanoma cell line, WM793. In addition, this membrane has also been cut into sections based on the molecular weights of the desired protein targets in order to allow for more than two antibodies to be examined on the same blot. For instance, the membrane analyzed in Figure 1 was cut in half in order to probe the lower portion of the membrane with a third antibody, total BIM (700 nm channel-red) without inferring with the detection of both total ERK1/2 and &#946;-Actin. The first panel of Figure 2 further demonstrates the two-color Western blot fluorescent detection of phospho-ERK1/2 (800 nm channel-green) and total ERK1/2 (700 nm channel-red) with overlapping phospho-ERK and total ERK signals displayed in yellow of WM793 melanoma cells treated in the presence and absence of the MEK1/2 inhibitor, PD0325901 (MEKi), which suppresses the ERK1/2 MAP kinase signaling pathway. Moreover, the 700 nm and 800 nm fluorescent images can also be converted to the standard black and white Western blot images for each antibody (Figures 1 and 2). In general, the majority of Western blot images produced by this protocol will result in high-quality images as exhibited in Figures 1 and 2. However, depending on the selectivity, specificity, and quality of the primary antibody, less than optimal images can result in a small portion of blots analyzed by this methodology. For example, as observed in the bottom two panels of Figure 2, the subpar quality of the phospho-BIM antibody in the WM793 melanoma cells treated with and without MEK inhibition resulted in a Western blot image with faint protein bands and extremely high membrane background.
Simultaneous infrared fluorescent imaging of two targets can also provide the opportunity for quantitative analysis of Western blots over a broad, linear dynamic range by efficiently and accurately detecting both strong and weak bands on the same membrane. Quantification of protein bands are calculated as an integrated intensity, which is proportional to the amount of fluorescent-labeled antibodies on the membrane and is independent of both size of the shape drawn around the band and resolution. This is based on the following criteria: 1) area of the shape; 2) number of pixels enclosed in the shape; 3) average intensity of the pixels selected as the background and; 4) total intensity of the pixels enclosed in the given shape10. Figure 3A shows the protein quantification of the Western blots illustrated in Figure 1. Quantification of total ERK1/2, &#946;-Actin, and total BIM exhibit a linear relationship between an increase in the integrated intensity of the fluorescent signal and the increase in protein concentration (Figure 3A). Moreover, Figure 3B is another example of how to quantify protein levels from fluorescent Western blots, which shows the protein quantification of phospho-ERK1/2 normalized to total ERK1/2 protein levels of WM793 melanoma cells treated with and without MEK inhibition from Figure 2. From this quantification, the phosphorylated ERK1/2 levels can be calculated as a percent of the control levels. In this case, phospho-ERK levels of melanoma cells treated with the MEK inhibitor were 0.6% of the control.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51149/51149fig1highres.jpg" src="/files/ftp_upload/51149/51149fig1.jpg" /> 
Figure 1. Western blot analysis using two-color infrared fluorescent protein detection. Lysates of two-fold serial dilutions of WM793 human-derived melanoma protein were separated using a NuPAGE Novex Bis-Tris gel with protein transferred onto PVDF membrane using the iBlot transfer apparatus. Membranes were blocked in Odyssey Blocking Buffer and probed with the following primary antibodies: rabbit anti-total ERK1/2, rabbit anti-total BIM, and mouse anti-&#946;-Actin. Antigen-antibody complexes were detected using fluorescent goat anti-rabbit IRDye 800 (green) or 680 (red) and goat anti-mouse IRDye 680 (red) secondary antibodies, respectively, and visualized with the LI-COR Odyssey Classic Infrared Imaging System. The first Western blot panel is the overlay of the simultaneous detection of total ERK1/2 (800 nm channel-green) and &#946;-Actin (700 nm channel-red) fluorescent signals. The second panel is the single-channel fluorescent detection of total BIM on the same membrane that was separated using a razor blade based on the molecular weights of these target proteins. The lower three Western blot panels are the black and white images of the separated single-channel fluorescent images.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51149/51149fig2highres.jpg" src="/files/ftp_upload/51149/51149fig2.jpg" /> 
Figure 2. An example of both a high- and poor-quality Western blot image using infrared fluorescence. WM793 melanoma cells treated with either DMSO (CTL) or 2 &#956;M of the MEK inhibitor, PD0325901 (MEKi), for 18 hr were analyzed as described in Figure 1. The membrane was probed with rabbit anti-phospho-ERK1/2 (Thr202/Tyr204), mouse anti-total ERK1/2, and mouse anti-phospho-BIM (Ser69) with antigen-antibody complexes detected with goat anti-rabbit IRDye 800 (green) and goat anti-mouse IRDye 680 (red). The first Western blot panel is the overlay of the simultaneous detection of both phospho-ERK1/2 (800 nm channel-green) and total ERK1/2 (700 nm channel-red) fluorescent signals. The second and third panels are the black and white images of the single-channel fluorescent images of phospho and total ERK1/2. The bottom two panels are the fluorescent and black and white images of phospho-BIM. These images represent an example of a subpar Western blot that can result with this method due to the substandard quality of the primary antibody.
<img alt="Figure 3" fo:content-width="2.75in" fo:src="/files/ftp_upload/51149/51149fig3highres.jpg" src="/files/ftp_upload/51149/51149fig3.jpg" /> 
Figure 3. Examples of protein quantification of the infrared fluorescent Western blots. A) Quantification of total ERK1/2, &#946;-Actin, and total BIM protein levels was determined by calculating the integrated intensity of each protein band. The integrated intensity is proportional to the amount of fluorescent-labeled secondary antibodies on the membrane. R-squared values were also used to evaluate the linear regression for each of the antibodies analyzed in Figure 1. B) Quantification of phospho-ERK1/2 normalized to total ERK1/2 protein levels from Figure 2 was calculated using the integrated intensity of the fluorescent signal and data is presented as a percent of the DMSO-control.

Watch this Scientific Journal Video about The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis at JoVE.com

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

the-fastest-western-town-contemporary-twist-on-classic-western-blot

Universidad San Sebastian

Research

JoVE Journal

Biology

27.1K Views.  University of California, San Francisco. This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

Video: The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere's disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain's protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) 
The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. 
In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.

This video shows how to surgically obstruct the guinea pig's endolymphatic duct to produce endolymphatic hydrops.

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.