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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li><li>Burnette, W. N. '. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate+polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+quantification+accuracy+for+Western+blots+Image+Analysis.">Improving quantification accuracy for Western blots Image Analysis.</a> , (2006).</li><li>Quan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species-mediated+endoplasmic+reticulum+stress+and+mitochondrial+dysfunction+contribute+to+cirsimartitin-induced+apoptosis+in+human+gallbladder+carcinoma+GBC-SD+cells.">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. , (2010).</li><li>O'Malley, D. e. r. v. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CytoBuster&trade; Protein Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71009</td>
<td><a href="http://www.emdbiosciences.com/cytobuster">www.emdbiosciences.com/cytobuster</a> <a href="http://www.merck4biosciences.com/cytobuster">www.merck4biosciences.com/cytobuster</a></td>
</tr>
<tr>
<td>PhosphoSafe&trade; Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71296</td>
<td><a href="http://www.emdbiosciences.com/phosphosafe">www.emdbiosciences.com/phosphosafe</a> <a href="http://www.merck4biosciences.com/phosphosafe">www.merck4biosciences.com/phosphosafe</a></td>
</tr>
<tr>
<td>Non-Interfering Protein Assay&trade; Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>488250</td>
<td><a href="http://www.emdbiosciences.com/noninterfering">www.emdbiosciences.com/noninterfering</a> <a href="http://www.merck4biosciences.com/noninterfering">www.merck4biosciences.com/noninterfering</a></td>
</tr>
<tr>
<td>BCA Protein Assay Kit </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71285</td>
<td><a href="http://www.emdbiosciences.com/BCA">www.emdbiosciences.com/BCA</a> <a href="http://www.merck4biosciences.com/BCA">www.merck4biosciences.com/BCA</a></td>
</tr>
<tr>
<td>4X SDS Sample Buffer </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70607</td>
<td><a href="http://www.emdbiosciences.com/4XSDS">www.emdbiosciences.com/4XSDS</a> <a href="http://www.merck4biosciences.com/4XSDS">www.merck4biosciences.com/4XSDS</a></td>
</tr>
<tr>
<td>Trail Mix&trade; Protein Markers</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70980</td>
<td><a href="http://www.emdbiosciences.com/trailmix">www.emdbiosciences.com/trailmix</a> <a href="http://www.merck4biosciences.com/trailmix">www.merck4biosciences.com/trailmix</a></td>
</tr>
<tr>
<td>ProteoExtract&trade; Protein Extraction Kits</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/proteoextract">www.emdbiosciences.com/proteoextract</a> <a href="http://www.merck4biosciences.com/proteoextract">www.merck4biosciences.com/proteoextract</a></td>
</tr>
<tr>
<td>OmniPur&reg; Products</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/omnipur">www.emdbiosciences.com/omnipur</a></td>
</tr>
<tr>
<td>RAPIDstain&trade; Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>553215</td>
<td><a href="http://www.emdbiosciences.com/rapidstain">www.emdbiosciences.com/rapidstain</a> <a href="http://www.merck4biosciences.com/rapidstain">www.merck4biosciences.com/rapidstain</a></td>
</tr>
<tr>
<td>RedAlert&trade; 10X Western Blot Stain</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71078</td>
<td><a href="http://www.emdbiosciences.com/redalert">www.emdbiosciences.com/redalert</a> <a href="http://www.merck4biosciences.com/redalert">www.merck4biosciences.com/redalert</a></td>
</tr>
<tr>
<td>SeaBlock&trade; Reagent, Salmon Plasma</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>558300</td>
<td><a href="http://www.emdbiosciences.com/seablock">www.emdbiosciences.com/seablock</a> <a href="http://www.merck4biosciences.com/seablock">www.merck4biosciences.com/seablock</a></td>
</tr>
<tr>
<td>BLOT-QuickBlocker Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>WB57</td>
<td><a href="http://www.emdbiosciences.com/quickblocker">www.emdbiosciences.com/quickblocker</a> <a href="http://www.merck4biosciences.com/quickblocker">www.merck4biosciences.com/quickblocker</a></td>
</tr>
<tr>
<td>Albumin, Bovine Serum, Fraction V</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>12660</td>
<td><a href="http://www.emdbiosciences.com/bovineserum">www.emdbiosciences.com/bovineserum</a> <a href="http://www.merck4biosciences.com/bovineserum">www.merck4biosciences.com/bovineserum</a></td>
</tr>
<tr>
<td>PBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524653</td>
<td><a href="http://www.emdbiosciences.com/PBST">www.emdbiosciences.com/PBST</a> <a href="http://www.merck4biosciences.com/PBST">www.merck4biosciences.com/PBST</a></td>
</tr>
<tr>
<td>TBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524753</td>
<td><a href="http://www.emdbiosciences.com/TBST">www.emdbiosciences.com/TBST</a> <a href="http://www.merck4biosciences.com/TBST">www.merck4biosciences.com/TBST</a></td>
</tr>
<tr>
<td>SignalBoost&trade; Immunoreaction Enhancer Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>407207</td>
<td><a href="http://www.emdbiosciences.com/signalboost">www.emdbiosciences.com/signalboost</a> <a href="http://www.merck4biosciences.com/signalboost">www.merck4biosciences.com/signalboost</a></td>
</tr>
<tr>
<td>RapidStep&trade; ECL Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>345818</td>
<td><a href="http://www.emdbiosciences.com/rapidstep">www.emdbiosciences.com/rapidstep</a> <a href="http://www.merck4biosciences.com/rapidstep">www.merck4biosciences.com/rapidstep</a></td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Western Blotting: Sample Preparation to Detection at JoVE.com

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.

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

western-blotting-sample-preparation-to-detection

Research

JoVE Journal

Biology

138.9K Views.  EMD Chemicals Inc.. Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Video: Western Blotting: Sample Preparation to Detection

MALDI Sample Preparation: the Ultra Thin Layer Method

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) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

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).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

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 ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

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.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

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 ...

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

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

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


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

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...