Name: Western Blotting: Sample Preparation to Detection (Video) | JoVE Journal
Uploaded: 2010-10-14T17:27:00.000+00:00
Duration: 7 min 45 s
Description: Learn Western blotting for protein detection in tissue samples using SDS-PAGE and membrane transfer. This method demonstrates how high-quality reagents and proper protocols enhance sensitivity and reliability.

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5 minutes

1. Sample Preparation
The first step in Western blotting is sample preparation. To prepare samples for running a gel cells and tissues need to be lysed to release the proteins of interest.
<ul>
	<li>A convenient, ready-to-use reagent that is versatile and easy to use for soluble protein extraction is CytoBuster or PhosphoSafe. These extraction buffers contain detergents optimized for efficient extraction of soluble proteins from mammalian and insect cells. The gentle, non-ionic composition of CytoBuster Protein Extraction Reagent enables isolation of functionally active endogenous or expressed proteins without a need for secondary treatment such as sonication or freeze/thaw. PhoshoSafe has the added benefit of containing four phosphatase inhibitors.
<ol>
	<li>Pellet the cells by low speed centrifugation (e.g. 5 min at 2500 x g), drain the cell pellet well.</li>
	<li>Resuspend the cells in CytoBuster Protein Extraction Reagent using 150 &mu;L per 106 cells (optimal amount of CytoBuster Protein Extraction Reagent may vary based on cell size).</li>
	<li>Incubate at room temperature for 5 min.</li>
	<li>Transfer to a suitable tube and spin for 5 min at 16,000 x g at 4&deg;C.</li>
	<li>Transfer cleared supernatant (cell extract) to a fresh tube and proceed with analysis.</li>
</ol>
</li>
	<li>For specialized protein extraction, we recommend using our high-quality ProteoExtract Kits. EMD has over a dozen kits for mitochondrial isolation, subcellular proteome extraction, transmembrane protein extraction, and many others. Please visit our Western blot landing page for further details.</li>
	<li>EMD also sells a wide variety of protease and phosphatase inhibitor cocktail sets. As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4&deg;C at all times and appropriate inhibitors are added fresh to the lysis buffer. Please visit our Western blot landing page for further details.</li>
</ul>
2. SDS-PAGE
The second step in Western blotting is protein separation. Proteins are separated based on molecular weight.
<ul>
	<li>You can make your own PAGE gel, however we will be using a commercially pre-cast gel. 
	<ol>
		<li>After preparing your sample, you are ready to determine the protein concentration. EMD has two kits for this to measure concentration, one being the Non-Interfering Protein Assay Kit and the other being the BCA Protein Assay Kit.</li>
		<li>Once the protein concentration is assessed we are ready to prepare our sample for loading the gel. Antibodies typically recognize a small portion of the protein of interest (referred to as the epitope) and this domain may reside within the 3D conformation of the protein. To enable access of the antibody to this portion it is necessary to unfold the protein.</li>
		<li>To denature, use a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95-100&deg;C for 5 minutes. An easy and convenient loading buffer to use is 4X Sample Buffer.</li>
 </ol>
 </li>
 <li>Load the first lane of the well with Trail Mix Protein Marker. This marker has three reference bands which allow you to monitor electrophoresis. When the gel is stained, 10 bands are visible ranging from 10-225 kDa.</li>
	<li>Fill the electrophoresis unit with running buffer. For PAGE gels, this is usually 1X Tris-glycine. For detailed buffer recipes, visit the Western blotting page. EMD offers high-quality buffers reagents with our OmniPur chemical line. Run the gel at 220V for 1 hour.</li>
	<li>Once the proteins have separated, stain the gel with Coomassie blue to ensure the proteins have migrated evenly and uniformly. RAPIDstain is an ultra-sensitive stain which is ready-to-use. No destaining is required.</li>
</ul>
3. Protein Transfer
Just as proteins with an electrical charge can be induced to travel through a gel in an electrical field, so can the proteins be transferred in an electrical field from the gel onto a membrane, being either PVDF or nitrocellulose.
<ul>
	<li>Today we will do a wet transfer. In wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane. The sandwich is submerged in transfer buffer to which an electrical field is applied. The negatively-charged proteins travel towards the positively-charged electrode, but the membrane stops them, binds them, and prevents them from continuing on.</li>
	<li>Two types of membranes are available: nitrocellulose and PVDF. Both work well. Today we will be using PVDF. PVDF membranes require soaking in methanol for a few minutes, followed by ice cold transfer buffer for 5 minutes. The gel also needs to equilibrate for a few minutes in ice cold transfer buffer. Not doing so may shrink the gel during transfer.</li>
	<li>The buffer for wet transfer is 1X Tris-glycine with 20% methanol. Detailed buffer preparations are available on our website.</li>
	<li>Once the transfer is complete, it's a good idea to visualize proteins to ensure even transfer with no air pockets. This can easily be done with RedAlert Western Blot Stain. This stain is ready-to-use and destaining can be done easily with water.</li>
</ul>
4. Antibodies and Accessory Reagents
<ul>
	<li>The first step in doing probing and antibody to antigen is to block the membrane. Blocking the membrane prevents non-specific background binding.</li>
	<li>Either non-fat milk or BSA can be used as a blocking solution. However, when using phospho-specific antibodies, it is not recommended to use milk as it will cause high background.</li>
	<li>EMD offers several high-quality blocking reagents, including SeaBlock, which is a non-mammalian blocking agent and BLOT QuickBlocker, which is prepared, ready-to-use blocking agent. We also have high-quality BSA (Fraction V) available.
<ol>
	<li>Incubate the membrane 1 hour at room temperature under gentle agitation.</li>
	<li>Wash three times in TBST or PBST after incubation. An easy and convenient way to make TBST or PBST is to use our tablets (PBS TWEEN Tablets/TBS TWEEN Tablets)</li>
</ol>
</li>
	<li>Once the membrane has been blocked, you are ready to incubate with the primary antibody. EMD has been offering a comprehensive portfolio of exceptional antibodies with the best antibody guarantee in the industry for over 30 years. Remember that with all EMD antibodies, we offer a full 100% no-risk guarantee. Purchase an antibody and use it for any species specificity or application you desire. If the antibody does not work to your satisfaction, we will provide a full credit to be used on any other product. For more details, please visit our Western blotting web page.
 <ol>
		<li>Today we will be incubating our primary antibody using solution 1 of SignalBoost. SignalBoost is a great product allowing your signal to be enhanced significantly. Simply incubate your primary antibody in Solution 1 and incubate for 1 hour or as you normally would. There is no need to add further blocking agents. Alternatively, you can use BSA or nonfat dry milk as a blocking agent.</li>
		<li>Wash with TBST. We make TBST using the ready-to-dissolve TBST tablet.</li>
		<li>Incubate your secondary antibody using solution 2 of SignalBoost. Incubate the secondary antibody in blocking buffer for 1 hour. Wash the membrane again several times with TBST.</li>
 </ol>
</li>
</ul>
5. Detection
For HRP conjugating secondary antibodies, ECL is traditionally used. An excellent ECL reagent we offer is RapidStep. The advantages of RapidStep are that no mixing of luminal or enhancer is required. Simply spray the membrane a few times and develop using x-ray film. RapidStep offers low pictogram sensitivity as well as emitting light for up to 2 hours after the addition of substrate.

<ol><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets%3A+procedure+and+some+applications%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li>
<li>Renart, J., Reiser, J., Stark, G. R., R, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera%3A+a+method+for+studying+antibody+specificity+and+antigen+structure%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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 (7), 3116-3120 (1979).</li>
<li>Burnette, W. N. 'W. estern <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((blotting%27%3A+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate+polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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, 195-203 (1981).</li>
<li>Ambroz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+quantification+accuracy+for+Western+blots+Image+Analysis%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Improving quantification accuracy for Western blots Image Analysis.</a> , (2006).</li>
<li>Quan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reactive+oxygen+species-mediated+endoplasmic+reticulum+stress+and+mitochondrial+dysfunction+contribute+to+cirsimartitin-induced+apoptosis+in+human+gallbladder+carcinoma+GBC-SD+cells%5BTitle%5D)+AND+%22Cancer+Lett+Article%22%5BJournal%5D)">Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimartitin-induced apoptosis in human gallbladder carcinoma GBC-SD cells.</a> Cancer Lett Article. , Forthcoming (2010).</li>
<li>O'Malley, D. ervia <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+in+colonic+corticotrophin-releasing+factor+receptors+in+the+maternally+separated+rat+model+of+irritable+bowel+syndrome%3A+Differential+effects+of+acute+psychological+and+physical+stressors%5BTitle%5D)+AND+%22Peptides%22%5BJournal%5D)">Alterations in colonic corticotrophin-releasing factor receptors in the maternally separated rat model of irritable bowel syndrome: Differential effects of acute psychological and physical stressors.</a> Peptides. 31 (4), 662-670 (2010).</li>
</ol>

The authors Anna Eslami and Jesse Lujan are employed by EMD Chemicals Inc that produces reagents and instruments used in this Article.

In this video presentation, we have highlighted the major steps in Western blotting which are sample preparation, SDS-PAGE, membrane blocking/probing with antibodies, and detection. For each step of the process, proper protocols and appropriate reagents should be used to attain high-quality results.

<table><tbody><tr><td>CytoBuster&amp;#8482; Protein Extraction Reagent</td><td>EMD Millipore</td><td>71009</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/cytobuster&quot;&gt;www.emdbiosciences.com/cytobuster&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/cytobuster&quot;&gt;www.merck4biosciences.com/cytobuster&lt;/a&gt;</td></tr><tr><td>PhosphoSafe&amp;#8482; Extraction Reagent</td><td>EMD Millipore</td><td>71296</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/phosphosafe&quot;&gt;www.emdbiosciences.com/phosphosafe&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/phosphosafe&quot;&gt;www.merck4biosciences.com/phosphosafe&lt;/a&gt;</td></tr><tr><td>Non-Interfering Protein Assay&amp;#8482; Kit</td><td>EMD Millipore</td><td>488250</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/noninterfering&quot;&gt;www.emdbiosciences.com/noninterfering&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/noninterfering&quot;&gt;www.merck4biosciences.com/noninterfering&lt;/a&gt;</td></tr><tr><td>BCA Protein Assay Kit </td><td>EMD Millipore</td><td>71285</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/BCA&quot;&gt;www.emdbiosciences.com/BCA&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/BCA&quot;&gt;www.merck4biosciences.com/BCA&lt;/a&gt;</td></tr><tr><td>4X SDS Sample Buffer </td><td>EMD Millipore</td><td>70607</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/4XSDS&quot;&gt;www.emdbiosciences.com/4XSDS&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/4XSDS&quot;&gt;www.merck4biosciences.com/4XSDS&lt;/a&gt;</td></tr><tr><td>Trail Mix&amp;#8482; Protein Markers</td><td>EMD Millipore</td><td>70980</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/trailmix&quot;&gt;www.emdbiosciences.com/trailmix&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/trailmix&quot;&gt;www.merck4biosciences.com/trailmix&lt;/a&gt;</td></tr><tr><td>Prote&amp;#8211;xtract&amp;#8482; Protein Extraction Kits</td><td>EMD Millipore</td><td></td><td>&lt;a href=&quot;http://www.emdbiosciences.com/prote&amp;#8211;xtract&quot;&gt;www.emdbiosciences.com/prote&amp;#8211;xtract&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/prote&amp;#8211;xtract&quot;&gt;www.merck4biosciences.com/prote&amp;#8211;xtract&lt;/a&gt;</td></tr><tr><td>OmniPur&amp;#174; Products</td><td>EMD Millipore</td><td></td><td>&lt;a href=&quot;http://www.emdbiosciences.com/omnipur&quot;&gt;www.emdbiosciences.com/omnipur&lt;/a&gt;</td></tr><tr><td>RAPIDstain&amp;#8482; Reagent</td><td>EMD Millipore</td><td>553215</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/rapidstain&quot;&gt;www.emdbiosciences.com/rapidstain&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/rapidstain&quot;&gt;www.merck4biosciences.com/rapidstain&lt;/a&gt;</td></tr><tr><td>RedAlert&amp;#8482; 10X Western Blot Stain</td><td>EMD Millipore</td><td>71078</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/redalert&quot;&gt;www.emdbiosciences.com/redalert&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/redalert&quot;&gt;www.merck4biosciences.com/redalert&lt;/a&gt;</td></tr><tr><td>SeaBlock&amp;#8482; Reagent, Salmon Plasma</td><td>EMD Millipore</td><td>558300</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/seablock&quot;&gt;www.emdbiosciences.com/seablock&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/seablock&quot;&gt;www.merck4biosciences.com/seablock&lt;/a&gt;</td></tr><tr><td>BLOT-QuickBlocker Reagent</td><td>EMD Millipore</td><td>WB57</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/quickblocker&quot;&gt;www.emdbiosciences.com/quickblocker&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/quickblocker&quot;&gt;www.merck4biosciences.com/quickblocker&lt;/a&gt;</td></tr><tr><td>Albumin, Bovine Serum, Fraction V</td><td>EMD Millipore</td><td>12660</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/bovineserum&quot;&gt;www.emdbiosciences.com/bovineserum&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/bovineserum&quot;&gt;www.merck4biosciences.com/bovineserum&lt;/a&gt;</td></tr><tr><td>PBS-TWEEN&amp;#174; Tablets</td><td>EMD Millipore</td><td>524653</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/PBST&quot;&gt;www.emdbiosciences.com/PBST&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/PBST&quot;&gt;www.merck4biosciences.com/PBST&lt;/a&gt;</td></tr><tr><td>TBS-TWEEN&amp;#174; Tablets</td><td>EMD Millipore</td><td>524753</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/TBST&quot;&gt;www.emdbiosciences.com/TBST&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/TBST&quot;&gt;www.merck4biosciences.com/TBST&lt;/a&gt;</td></tr><tr><td>SignalBoost&amp;#8482; Immunoreaction Enhancer Kit</td><td>EMD Millipore</td><td>407207</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/signalboost&quot;&gt;www.emdbiosciences.com/signalboost&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/signalboost&quot;&gt;www.merck4biosciences.com/signalboost&lt;/a&gt;</td></tr><tr><td>RapidStep&amp;#8482; ECL Reagent</td><td>EMD Millipore</td><td>345818</td><td>&lt;a href=&quot;http://www.emdbiosciences.com/rapidstep&quot;&gt;www.emdbiosciences.com/rapidstep&lt;/a&gt;&lt;br/&gt;&lt;a href=&quot;http://www.merck4biosciences.com/rapidstep&quot;&gt;www.merck4biosciences.com/rapidstep&lt;/a&gt;</td></tr></tbody></table>

western blotting: sample preparation to detection

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 overall goal of this procedure is to demonstrate the ease of performing a Western blot when using appropriate products. Following sample preparation, proteins are separated based on size and then transferred to a solid phase. The proteins are then probed with antibodies and detected the use of proper protocols and high quality cost-effective reagents produce enhanced western blotting results.Hi, I'm Jesse Luhan, r and d scientist at EMD Chemicals in San Diego, California. EMD is an affiliate of Merck, KGAA in Dharm stat Germany. Today we're gonna show you a western blotting procedure.So let's get started. The first step in western blotting is sample preparation. Begin sample preparation by pelleting the cells using low speed centrifugation such as five minutes at 2, 500 times gravity.Following centrifugation, drain the cell pellets. Well resuspend the cells in cyto buster protein extraction reagent using 150 microliters per 10 to the six cells. Incubate the sample at room temperature for five minutes.Next, spin the sample for five minutes at 16, 000 times gravity at four degrees Celsius following centrifugation. Transfer the cleared SNA to a fresh tube and proceed with analysis. After the protein concentration of the sample is assessed, prepare the sample for loading the gel to denature the protein.Use a sample buffer containing an onic denaturing detergent such as sodium doil sulfate or SDS. Boil the mixture at 95 to 100 degrees Celsius for five minutes. Using a commercially available precast gel proteins can quickly be separated based on molecular weight.Fill the electrophoresis unit with running buffer, which can be made from reagents offered through the Omni PU chemical line. Load the first lane of the well with trail mix protein marker. This marker has three reference bands which allow for monitoring during electrophoresis.Once the gel is stained, 10 bands are visible, ranging from 10 to 225 kilo Daltons. Load the denatured sample into the remaining wells. Run the gel at 220 volts for one hour.Once the proteins have separated, stain the gel with kumasi blue to ensure that the proteins have migrated evenly and uniformly using rapid stain and ultrasensitive stain, which is ready to use no DS.Staining is required just as proteins with an electrical charge can be induced to travel through a gel in an electrical field, so the proteins can be transferred in an electrical field from the gel onto a membrane such as NITROCELLULOSE or PVDF. When using PVDF membranes, soak the membrane in methanol for a few minutes, followed by a equilibration in ice cold transfer buffer for five minutes. Also soak the gel for a few minutes in ice cold transfer buffer E equilibration prevents shrinkage of the gel during transfer.For a wet transfer, assemble a sandwich of the gel and membrane between sponge and paper. Then clamp the assembly together tightly after ensuring no air bubbles have formed between the gel and membrane. Submerge the sandwich in transfer buffer and apply an electrical field.The negatively charged proteins will travel towards the positively charged electrode. The membrane then stops them, binds them, and prevents them from continuing on. Once the transfer is complete, visualize proteins to ensure even transfer with no air pockets.This can easily be done with red alert Western blot stain. This stain is ready to use and des staining can be done easily with water. This is the gel once it has been distained by washing in water before the membrane can be proved with antibody, the membrane is blocked.To prevent nonspecific binding, block the membrane with high quality blocking reagents. To avoid an increased background by incubating it in blocking solution for one hour at room temperature under gentle agitation, then wash the membrane three times in TBST or PBST after incubation. These buffers can be easily and conveniently made using TBS tween tablets and PBS tween tablets respectively.Once the membrane has been blocked and washed, it can be incubated with the primary antibody. Apply the primary antibody using solution, one of signal boost, which allows the signal to be enhanced significantly. Prepare the primary antibody in solution one and incubate the membrane for one hour.There is no need to add further blocking agents following incubation. Wash the membrane with TBST. Next, incubate the membrane with the secondary antibody diluted in solution two of signal boost for one hour.Finally, wash the membrane again several times with TBST for HRP conjugated. Secondary antibodies detection by enhanced chemiluminescence or ECL is traditionally used here. The ECL reagent rapid step is used as it requires no mixing of luminol or enhancer.Simply spray the membrane a few times, incubate for two minutes, and develop it using x-ray film. Rapid Step offers low picogram sensitivity as well as lighter mission for up to two hours after the addition of substrate. Trail mix protein markers allows for protein tracking during electrophoresis when compared with standard Western blot protocols.Signal Boost allows for greater dynamic range and sensitivity. Finally, superior sensitivity can be obtained by using Rapid step instead of traditional ECL reagents. We've just shown you how to perform Western blinding efficiently and cost-effectively with a comprehensive range of products.Remember the quality of your results rely on the quality of your reagents. That's it. Thank you for watching and good luck with your experiments.

western-blotting-sample-preparation-to-detection

Research

JoVE Journal

Biology

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

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

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.

Immunoblot Analysis

This video demonstrates a quantitative dot blot, or QDB, technique for the absolute quantification of proteins of interest in a tissue sample. The proteins in the sample are immobilized on the nitrocellulose membrane inside the wells of a QDB plate. Utilizing target protein-specific antibodies labeled with a peroxidase enzyme, a chemiluminescent substrate is oxidized to produce light, which is measured to determine the protein concentration of the sample.

Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

The video demonstrates a total protein normalization-based western blotting technique for quantifying the target protein expression in a sample. By utilizing the total protein staining of the sample, a ratiometric analysis is performed to determine the relative expression level of the target protein.

Quantitative Western Blotting to Estimate Protein Expression Levels

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.

Chemiluminescent Western Blot Assay for Protein Processing

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.

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

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.

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Sample Preparation: the Ultra Thin Layer Method

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Learn Western blotting for protein detection in tissue samples using SDS-PAGE and membrane transfer. This method demonstrates how high-quality reagents and proper protocols enhance sensitivity and reliability.

Western Blotting: Sample Preparation to Detection

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