This work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health. S.H. was supported by Japan Public-Private Partnership Student Study Abroad Program, and H.N. and K.Y. were by Valor and V Drug Overseas Training Scholarship.

1. SDS-PAGE and transfer to a PVDF membrane
<ol>
	<li>Perform SDS-PAGE to separate proteins based on relative size. 
	NOTE: Any commercial or home-made gel system is fine. Please follow the manufacturer&#8217;s protocol.</li>
	<li>Transfer separated proteins from a gel onto a PVDF membrane. 
	NOTE: Common transfer methods (semi-dry or wet transfer) are suitable. Please follow the manufacturer&#8217;s protocol.</li>
</ol>
2. Blocking of PVDF membranes
<ol>
	<li>Incubate the membranes ...

<ol>
	<li>MacPhee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+considerations+for+improving+Western+blot+analysis.">Methodological considerations for improving Western blot analysis.</a> Journal of Pharmacol Toxicology Methods. 61 (2), 171-177 (2010).</li><li>Janes, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+critical+factors+for+quantitative+immunoblotting.">An analysis of critical factors for quantitative immunoblotting.</a> Science Signaling. 8 (371), (2015).</li><li>Schuck, P., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mass+transport+limitation+and+surface+heterogeneity+in+the+biophysical+characterization+of+macromolecular+binding+processes+by+SPR+biosensing.">The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.</a> Methods in Molecular Biology. 627, 15-54 (2010).</li><li>Li, J., Zrazhevskiy, P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eliminating+Size-Associated+Diffusion+Constraints+for+Rapid+On-Surface+Bioassays+with+Nanoparticle+Probes.">Eliminating Size-Associated Diffusion Constraints for Rapid On-Surface Bioassays with Nanoparticle Probes.</a> Small. 12 (8), 1035-1043 (2016).</li><li>Higashi, 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=Old+but+not+Obsolete:+An+Enhanced+High+Speed+Immunoblot.">Old but not Obsolete: An Enhanced High Speed Immunoblot.</a> Journal of Biochemistry. 168 (1), 15-22 (2020).</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> Proceedings of the National Academy of Sciences of the United States of America. 76, 4350-4354 (1979).</li><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> Analytical Biochemistry. 112 (2), 195-203 (1981).</li><li>Mishra, M., Tiwari, S., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification+and+analysis:+next+generation+Western+blotting+techniques.">Protein purification and analysis: next generation Western blotting techniques.</a> Expert Review of Proteomics. 14 (11), 1037-1053 (2017).</li><li>Ciaccio, M. F., Wagner, J. P., Chuu, C. P., Lauffenburger, D. A., Jones, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+analysis+of+EGF+receptor+signaling+dynamics+with+microwestern+arrays.">Systems analysis of EGF receptor signaling dynamics with microwestern arrays.</a> Nature Methods. 7 (2), 148-155 (2010).</li><li>Treindl, F., 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+bead-based+western+for+high-throughput+cellular+signal+transduction+analyses.">A bead-based western for high-throughput cellular signal transduction analyses.</a> Nature Communications. 7, 12852 (2016).</li><li>Sajjad, S., Do, M. T., Shin, H. S., Yoon, T. S., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+western+blot+assay+by+rotational+cyclic+draining+and+replenishing+procedure.">Rapid and efficient western blot assay by rotational cyclic draining and replenishing procedure.</a> Electrophoresis. 39 (23), 2974-2978 (2018).</li><li>Kurien, B. T., Scofield, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+other+notable+protein+detection+methods+on+blots.">A brief review of other notable protein detection methods on blots.</a> Methods in Molecular Biology. 536, 557-571 (2009).</li><li>Nagase, H., 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=Reliable+and+Sensitive+Detection+of+Glycosaminoglycan+Chains+with+Immunoblots.">Reliable and Sensitive Detection of Glycosaminoglycan Chains with Immunoblots.</a> Glycobiology. , (2020).</li></ol>

Western blot is a widely used analytical technique to detect specific proteins that was developed ~40 years ago7,8. Since then, the technique has continued to evolve, with subsequent innovations improving the sensitivity, speed, and quantitation of the technique2,9,10,11,12,<sup class="xref"...

A western blot (also known as an immunoblot) is a powerful and fundamental technique across a broad range of scientific and clinical disciplines. This technique is used to examine the presence, relative abundance, relative molecular mass, and post-translational modifications of proteins1. Combined with digital image analysis, this method can reliably analyze the abundance of proteins and protein modifications2. Although western blot is performed routinely, it is a time-consuming and labor intensive method. Long incubations of the antibody with membranes are required. Here, we describe a modification of the incubation method that overcomes this limitation without sacrificing sensitivity.
During incubation of the membrane, antibodies float in solution while antigens are immobilized on the membrane. Because of their high affinity, the rate of antibody binding to the antigen is faster than the diffusion of antibodies from the bulk solution to the membrane. This creates a low concentration &#8220;depletion layer&#8221; (Figure 1). It may take hours for more distant antibodies to reach the membrane via passive diffusion, which is the main factor responsible for long incubation times in this technique. This effect is called the mass transport limitation (MTL)3. It has been proposed that repetitive draining and replenishing the antibody-containing solution may disrupt the depletion layer and overcome MTL4. Here, we have devised a unique technique that implements this cyclic draining and replenishing (CDR) concept to diminish the effect of MTL in a traditional western blotting protocol and remarkedly shorten the required incubation period.
In order to maintain the detection sensitivity with a short incubation time, we made use of an immunoreaction enhancing technology that accelerates the antigen-antibody reaction, thereby improving the signal-to-noise ratio5. Many commercially available immunoreaction enhancing agents (IRE) consist of 2 components (Solution 1 and 2, see Table of Materials). The proprietary composition or mechanism of action of IRE has not been disclosed, but we have previously found that IRE decreased the dissociation constant between the antigen and antibody in solid phase binding assays, indicating increased affinity is, at least in part, responsible for the enhancing effect of IRE6. The combination of CDR with IRE yields an ultra-high-speed western blotting protocol that reduces the entire procedure time without sacrificing sensitivity.

<table>
	<tbody>
		<tr>
			<td>14 mL Round Bottom High Clarity PP Test Tube, Graduated, with Snap Cap</td>
			<td>Falcon</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Apollo Transparency Film for Laser Printers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any kinds of Laser Printer Transparency Film are fine.</td>
		</tr>
		<tr>
			<td>Azure Imaging System 600</td>
			<td>Azure Biosystems</td>
			<td>Azure 600</td>
			<td>Any kind of Image Acquiring Sytem is fine for chemiluminescent and/or fluorescent detection.</td>
		</tr>
		<tr>
			<td>Can Get Signal Immunoreaction Enhancer Solution</td>
			<td>TOYOBO</td>
			<td>NKB-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CARNATION Instant Nonfat Dry Milk</td>
			<td>Carnation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-mouse IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from sheep</td>
			<td>GE Healthcare</td>
			<td>NA9310V</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-rabbit IgG, Horseradish peroxidase linked F(ab&#8217;)2 fragment from donkey</td>
			<td>GE Healthcare</td>
			<td>NA9340V</td>
			<td></td>
		</tr>
		<tr>
			<td>HYBAID Micro-4</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any hybridization oven is fine as long as the tubes are rotated evenly at a horizontal position.</td>
		</tr>
		<tr>
			<td>Immobilon-P PVDF Membrane, 0.45 &#181;m pore size</td>
			<td>Millipore Sigma</td>
			<td>IPVH304F0</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-68070</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LICOR</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>KPL LumiGLO Reserve Chemiluminescent Substrate</td>
			<td>seracare</td>
			<td>5430-0049</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#223; actin antibody</td>
			<td>Millipore Sigma</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (PBS)</td>
			<td>LICOR</td>
			<td>927-40100</td>
			<td>Blocking Buffer for fluorescent detection</td>
		</tr>
		<tr>
			<td>OXO Salad Spinner</td>
			<td>OXO</td>
			<td>32480V2B</td>
			<td>Any salad spinner is fine as long as the PVDF membranes are rinsed vigorously without tear.</td>
		</tr>
		<tr>
			<td>Parafilm Sealing Film</td>
			<td>Bemis</td>
			<td>Parafilm M PM996</td>
			<td>semi-transparent flexible film</td>
		</tr>
		<tr>
			<td>Polyethylene Flat-Top Screw Caps for 50 mL Conical Bottom Centrifuge Tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Rings to hold 14 ml tube in the center of 50 ml tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared in a machine shop</td>
		</tr>
		<tr>
			<td>Rabbit anti-6X His tag antibody</td>
			<td>Abcam</td>
			<td>ab9108</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Millipore Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

ultra-high-speed western blot using immunoreaction enhancing technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

This example illustrates the effectiveness of the CDR method together with immunoenhancing technology on western blot. Static incubation was performed on a semi-transparent flexible film where PVDF membranes were incubated with the antibody solution, whereas CDR incubation was in a hybridization oven by rotating tubes that contained the membranes and the antibody solution (Movie). Figure 2 showed quantities of antigen and antibody, respectively, necessary for chemiluminescent detection on we...

Watch this Scientific Journal Video about Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology at JoVE.com

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Western blotting is an established method that detects antigens on a transferred membrane and is commonly used in biomedical research, but it takes hours or days to complete the entire procedure. Using the combination of immuno-enhancing technology with cyclic draining and replenishing, once you've got a block membrane, the entire procedure can be accomplished in as little as 20 minutes without sacrificing sensitivity. Because Western blotting is common to many different research areas, it has broad applicability to speed up research.The successful use of this protocol relies on the optimization of antibody dilution with immunoreaction enhancing reagent and incubation time. Begin by incubating the PVDF membranes in appropriate blocking buffers for one hour with agitation at room temperature. While the membranes are incubating, prepare 10%immunoreaction enhancing agent one solution or IRE1 with distilled water and vortex it well, then dilute the primary antibody with 10%IRE1, mixing it gently and thoroughly.Pick up the PVDF membrane with tweezers and briefly drain the blocking solution. Insert the membrane into a conical tube with primary antibody, ensuring that the entire membrane adheres to the wall of the tube and that the side of the membrane that was originally in contact with the gel is facing inward. Cap the tube tightly.Insert the tube into a glass bottle for the hybridization oven and turn on the oven. Incubate the membrane with six RPM rotation for at least five minutes. Stop the rotation and carefully remove the PVDF membrane from the tube with tweezers.Place the membrane into a container with 50 milliliters of PBST and rinse it, then rinse it in a container with distilled water until no bubbles appear, which will remove the majority of the antibody. Transfer the membrane to a salad spinner that contains 250 milliliters of PBST. Place the strainer basket in the spinner and ensure that the lid has been put on securely, then activate the spinner and run it for 20 to 30 seconds.Discard the solution and briefly rinse the inside of the spinner with distilled water. Add 250 milliliters of PBST into the spinner and repeat the spin, then incubate the membrane with the secondary antibodies and wash it again as described in the text manuscript. Place a piece of semi-transparent flexible film on a flat surface.Mix two components of the chemiluminescent substrate in a five milliliter tube and immediately transfer 1.5 milliliters of the mixed substrate to the semi-transparent film. Place the PVDF membrane on the solution and transfer the rest of the substrate solution onto the membrane. Incubate the PVDF membrane with the mixed substrate for one minute.Pick up the PVDF membrane with tweezers and drain the substrate solution briefly. Sandwich the membrane between a pair of transparency films and image it under chemiluminescent mode. The quantities of antigen and antibody respectively necessary for chemiluminescent detection of Western blot are shown here.In both static and CDR incubations, usage of IRE solutions increased the sensitivity. Similarly, the minimum concentration of anti-6s His tag antibody was lowered from 100 to 50 nanograms per milliliter under static conditions and from 100 to 12.5 nanograms per milliliter under CDR conditions. CDR incubation for either 5 or 10 minutes dramatically lowered the detection limit compared to static incubation.Furthermore, the combination of CDR with IRE revealed superior sensitivity on the Western blot, even within the extremely short incubation period. Successful application of this method to a Western blot with fluorescent detection is demonstrated here. Fluorescent detection in contrast to chemiluminescent detection has the unique ability to detect multiple targets on the same blot at the same time without stripping and reprobing antibodies.His-tagged alkaline phosphatase and beta-actin in the cell lysates were simultaneously detected using a two-color fluorophore. CDR incubation with IRE not only accelerated the detection, but also increased the sensitivity. During the incubation, the membrane should stay attached to the wall of the tube.The tube should be rotated horizontally so that solution washes continually over the membrane surface. This protocol significantly accelerated our research productivity, which strongly depends on Western blotting procedures.

ultra-high-speed-western-blot-using-immunoreaction-enhancing

Research

JoVE Journal

Biochemistry

5.7K visualizações.  National Institutes of Health. Western blotting is an established method that detects antigens on a transferred membrane and is commonly used in biomedical research, but it takes hours or days to complete the entire procedure. Using the combination of immuno-enhancing technology with cyclic draining and replenishing, once you've got a block membrane, the entire procedure can be accomplished in as little as 20 minutes without sacrificing sensitivity. Because Western blotting is common to many different research areas, it ha...