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 in appropriate blocking buffers for 1 h with agitation at room temperature. 
	NOTE: Depending on the primary antibody and detection system, an appropriate blocking buffer should be selected. For example, the most typical blockers are BSA, nonfat dry milk, casein, and commercial synthetic polymers. PBS and/or TBS with 0.1% Tween20 are the most commonly used buffers. This blocking step may be done overnight at 4 &#730;C.</li>
</ol>
3. Incubation with primary antibody
<ol>
	<li>Prepare 10% immunoreaction enhancing agent-1 solution (IRE-1) with distilled water. Vortex well.</li>
	<li>Prepare the diluted primary antibody with 10% IRE-1. Mix it gently and thoroughly.</li>
	<li>If using a larger membrane (4 cm x 8 cm &#8211; 8 cm x 8 cm), add 8 mL of the antibody solution into 50 mL conical centrifuge tubes. If using a smaller membrane (2 cm x 8 cm &#8211; 4 cm x 8 cm), add 3 mL of the antibody solution into 14 mL round-bottom polypropylene tube (Table of Materials).</li>
	<li>Pick up the PVDF membrane with tweezers and drain the blocking solution briefly. Insert the PVDF membrane into this tube with gloved fingers and ensure that the entire membrane adheres to the wall of the tube. When the PVDF membrane is inserted into a tube, face the &#8220;protein side&#8221; of the membrane that was originally in contact with the gel inward. Close the cap tightly.</li>
	<li>Insert the 50 mL tube into a glass bottle for the hybridization oven. 
	NOTE: When 14 mL tubes are used for this incubation, insert a 14 mL tube into the 50 mL tube, using a pair of plastic ring holders to keep the inner tube in the center of the 50 mL tube.</li>
	<li>Turn on the hybridization oven.</li>
	<li>Incubate the membrane with 6 rpm rotation for at least 5 min. 
	NOTE: Make sure that the membrane adheres to the wall at all times and that the (inner) tube is horizontal to cover the membrane with the antibody solution evenly. Calibrate the dilution of the antibody and incubation time prior to the actual experiment.</li>
</ol>
4. Membrane wash
<ol>
	<li>Stop the rotation.</li>
	<li>Carefully remove the PVDF membrane from the tube with tweezers, and place the membrane into a container with 50 mL of PBS-T.</li>
	<li>Rinse the membrane briefly with PBS-T.</li>
	<li>Rinse the membrane in the container with distilled water until no bubbles appear. This is to remove the majority of the antibody.</li>
	<li>Transfer the PVDF membrane from the container to the salad spinner that contains 250 mL of PBS-T.</li>
	<li>Place the strainer basket in the spinner. Ensure that the lid has been put on securely.</li>
	<li>Activate the spinner and run it for about 20-30 s.</li>
	<li>Discard the solution and briefly rinse the inside of the spinner with distilled water.</li>
	<li>Add 250 mL of PBS-T into the spinner again.</li>
	<li>Repeat steps 4.6 and 4.7.</li>
</ol>
5. Incubation with the secondary antibody
<ol>
	<li>Prepare 10% immunoreaction enhancing agent-2 solution (IRE-2) with distilled water. Vortex well.</li>
	<li>Prepare the diluted secondary antibody with 10% IRE-2. Mix it gently and thoroughly.</li>
	<li>Select the volume of the antibody solution and tube as stated in step 3.3.</li>
	<li>Pick up the PVDF membrane with tweezers and drain the solution briefly. Insert the PVDF membrane into the tube as stated in step 3.4.</li>
	<li>Insert the 50 mL tube into a glass bottle for the hybridization oven.</li>
	<li>Turn on the hybridization oven.</li>
	<li>Incubate the membrane with 6 rpm rotation for at least 5 min. Make sure that the membrane adheres to the wall at all times and that the (inner) tube is horizontal to evenly cover the membrane with the antibody solution. 
	NOTE: Calibrate the incubation time prior to the actual experiment. When the secondary antibody is fluorescently conjugated, perform the incubation in the dark.</li>
</ol>
6. Membrane wash
<ol>
	<li>Follow steps 4.1 to 4.10.</li>
</ol>
7. Image acquisition
<ol>
	<li>Chemiluminescent detection
	<ol>
		<li>Place a piece of semi-transplant flexible film (10 cm x 15 cm) on a flat surface.</li>
		<li>Mix 2 components of the chemiluminescent substrate in a 5&#160;mL tube.</li>
		<li>Immediately transfer 1.5 mL of the mixed substrate to the semi-transplant flexible film and place the PVDF membrane upon it. Then, transfer the rest of the substrate solution onto the membrane.</li>
		<li>Incubate the PVDF membrane with the mixed substrate on a semi-transparent flexible film for 1 min.</li>
		<li>Pick up the PVDF membrane with tweezers and drain the substrate solution briefly.</li>
		<li>Sandwich the membrane between a pair of transparency films.</li>
		<li>Acquire the image under chemiluminescent mode.</li>
	</ol>
	</li>
	<li>Fluorescent detection
	<ol>
		<li>Pick up the PVDF membrane with tweezers and drain the&#160;solution briefly.</li>
		<li>Sandwich the membrane between a pair of transparency films.</li>
		<li>Acquire the image under fluorescent mode. 
		NOTE: Steps 7.1 and 7.2 should be performed in close proximity to the imaging machine to ensure maximum sensitivity.</li>
		<li>When the background signal is high, perform a washing step in a container with 80-100 mL of PBS-T: 10 min rinse 3 times for the primary antibody and 5 min rinse 6 times for the secondary antibody. 
		NOTE: Both primary and secondary antibodies diluted with 10% IRE solutions can be used up to 8-10 times without loss of sensitivity6. The solution should be kept at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
</ol>

<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>

The authors have declared no competing interests directly relevant to the contents of this article.

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 technique2,9,10,11,12,13. The enhanced ultra-high-speed western blotting protocol presented here makes several substantial enhancements to the existing technique. We reduce incubation time without sacrificing sensitivity by lowering the detection threshold with IRE. No special or expensive equipment is needed: rotating a tube in a hybridization oven is sufficient to overcome MTL. The key innovation is that CDR effectively eliminates the depletion layer in the assay. Although a roller-culture apparatus is commonly employed to reduce the volume of solution, the potential of this method to overcome MTL and reduce incubation times has not been illustrated. Because of the drastic reduction of the overall time for the entire procedure (Figure 4), much higher quantities of western blot data per day can be obtained when the blocked membranes are in one&#8217;s hand, which is almost impossible to do so with a traditional western blotting protocol. It should be noted that incubation of the membrane on a rocking platform shaker is equivalent to static incubation4,6. Rinsing PVDF membranes in a household commercial salad spinner with a large volume of washing solution also decreases the duration of the procedure (Figure 4).
The successful use of this protocol relies on the optimization of the antibody dilution with IRE solution and incubation time. A wide range of antibody titration is recommended at the beginning of the assays since CDR in conjunction with IRE increases the sensitivity substantially (Figure 2). Incubation time is another factor to be considered. When one has good western blot results with 1 h incubation with primary antibodies under static conditions, incubation time could be reduced to 5-10 min under CDR conditions in general. When overnight incubation is needed with primary antibodies for example, a slightly longer CDR incubation time (30 min - 6 h) should be evaluated. Dilution and incubation time with secondary antibodies are also critical. Incubations greater than 30 min under CDR conditions usually give higher background. Thus, careful titration of secondary antibodies is required with up to 30 min of CDR incubation. When the background signal is high after careful titration of antibodies, extensive washing steps (10 min rinse 3 times for the primary antibody and 5 min rinse 6 times for the secondary antibody in a container with 80-100 mL of washing solution) should be performed.
The quality of the western blot data often depends on the antibodies used. When we had a difficult antibody used for western blot, protein markers sometimes showed substantial signals even after careful antibody titration and extensive washing. Since we noticed that the CDR method with IRE tends to increase the non-specific binding, addition of blocking reagents (such as skim milk and others) into antibody diluents may improve the signal-to-noise ratio14. It is a time-consuming screening step, but it is worth trying.
The western blot has become important for quantitative applications2. A semi-quantitation can be performed with chemiluminescent detection. The procedure presented here is applicable to stripping and re-probing to this end6. Because of a wider dynamic range, fluorescent detection is the best option. With this protocol, fluorescent detection provides a linear dynamic range for quantitative analysis6. It is worth mentioning that CDR incubation time with fluorescent detection seems longer than that with chemiluminescent detection.
The western blot has a wide array of applications and remains a universal method to study protein abundance, protein-protein interactions, and post-translational modifications. For instance, anti-carbohydrate antibodies can be easily used for western blot with this protocol14.&#160;Since a variety of carbohydrate moieties expressed on the outer surface of viral, bacterial, and fugus are pathogen-specific, they are invaluable targets for pathogen recognition and diagnosis of infectious diseases. Thus, the application of this simple protocol could impact many areas of investigation, shaving hours of waiting time not only for experiments but also clinical immunodiagnostics that rely on western blotting technology.

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&amp;rsquo;)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&amp;rsquo;)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 &amp;micro;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-&amp;szlig; 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 western blots. In both static and CDR incubations, usage of IRE solution increased the sensitivity: IRE reduced the lowest quantity of cell lysates (antigen) needed to detect &#223;-actin from 2.2 &#181;g to 1.1 &#181;g under static conditions and further reduced from 1.1 &#181;g to 0.275 &#181;g using CDR (Figure 2A). Similarly, the minimum concentration of anti-6x His tag antibody was lowered from 100 ng/mL to 50 ng/mL under static conditions and from 100 ng/mL to 12.5 ng/mL under CDR condition (Figure 2B). CDR incubation for either 5 min or 10 min dramatically lowered the detection limit compared to static incubation (60 min with primary and 30 min with secondary antibodies). Finally, the combination of CDR with IRE revealed superior sensitivity on the western blot (0.275 &#181;g of cell lysates for &#223;-actin detection and 12.5 ng/mL of anti-6x His tag antibody) even within this extremely short incubation period.
The second example demonstrates the successful application of this method to a western blot with fluorescent detection (Figure 3). 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/re-probing antibodies. (His)6&#160;tagged&#160;AP and &#223;-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, resulting in unmasking the degradation of (His)6 tagged AP (Figure 3A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61657/61657fig01.jpg" /> 
Figure 1: Schematic illustration of the cyclic draining-replenishing (CDR) method. 
(A) A depletion layer near the membrane rapidly forms under static conditions when a probe has a high affinity for the antigen but a limited ability to diffuse through the solution. Thus, travel of the probe to the membrane becomes a rate limiting step and long incubation times compensate for mass transfer limitation. (B) CDR method by rotating the tube while the membrane adheres to the wall eliminates the depletion layer and replenishes the same probe solution to keep the probe concentration near the membrane constant. This figure has been modified from Higashi et al.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61657/61657fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61657/61657fig02.jpg" /> 
Figure 2: (A) Different amounts of 293 cell lysates (1:2 serial dilutions from 8.8 &#181;g/lane) were separated by SDS-PAGE, followed by transfer to a PVDF membrane and blocking with 5% skim milk in PBS-T. The blot was probed with mouse anti-&#223;-actin antibody (1:3,000 dilution). (B) After separation of conditioned media derived from transfected 293 cells with pAPTAG5 (GenHunter) containing the secreted (His)6 tagged alkaline phosphatase (AP, 8 x 10-14 mol/lane), proteins were transferred to PVDF membranes. Each membrane was subjected to western blot with different concentrations of anti-6x His tag antibody (1:2 serial dilutions from 400 ng/mL). The membranes were imaged as a single image and dotted lines indicate the border of individual membranes. This figure has been modified from Higashi et al.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61657/61657fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61657/61657fig03.jpg" /> 
Figure 3: Simultaneous detection of multiple targets on a western blot using fluorescent detection and CDR in conjunction with IRE. 
Different amounts of lysates (1:2 serial dilutions from 10 &#181;g/lane) from 293 cells transfected with pAPTAG5 were separated and transferred to a PVDF membrane. After blocking with the Blocking Buffer for fluorescent detection (BFD) for 1 h, the blot was probed with anti-6X His tag and anti &#223;-actin antibodies, followed by IRDye 800CW goat anti-rabbit IgG and IRDye 680RD goat anti-mouse IgG. A: antibodies diluted with 10% IRE solution under CDR condition, B: antibodies diluted with BFD containing 0.1% Tween20 under static condition. Because of higher sensitivity with CDR and IRE combination, the degradation of (His)6 tagged AP was seen. This figure has been modified from Higashi et al.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61657/61657fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61657/61657fig04.jpg" /> 
Figure 4: Schematic illustration of an enhanced and ultra-high speed CDR western blot compared to a traditional static method. 
This figure has been modified from Higashi et al.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61657/61657fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

5.9K Views.  National Institutes of Health. Een ultrasnelle westerse blotting-techniek wordt ontwikkeld door de kinetiek van antigeen-antilichaambinding te verbeteren door middel van cyclische draining- en replenishing (CDR)-technologie in combinatie met een immunoreactieverhogend middel.

Video: Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Biology

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

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.

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 ...

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 ...

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

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Advanced Biology

Immunology

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

Basic Biology

Basic Methods in Cellular and Molecular Biology

Chemiluminescent Western Blot Assay for Protein Processing

Source: Hillert-Richter, L. K. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62842">Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex.</a> J. Vis. Exp. (2021).
This video describes the chemiluminescence-based western blotting of immunoprecipitated lysates. The technique helps determine protein processing and activation. The chemiluminescent signal detected is proportional to the level of protein processing during its activation.

Source: Hillert-Richter, L. K. et al., Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex. J. ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

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