Total Protein Normalization

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Western Blot Normalization Methods

A quantitative western blot accurately measures changes in protein expression by making relative comparisons between different treatments.

A properly designed quantitative western blotting experiment ensures that:

  • The amount of protein loaded is proportional to the signal intensity
  • All quantified targets' signal intensities fall within the linear range of the imaging system
  • Common blot-to-blot variations are accurately assessed by an internal loading control

Western blot normalization allows you to faithfully compare changes in protein expression by establishing the baseline needed to correct against common errors such as inconsistent sample preparation, pipetting error, and uneven protein transfer. Proper western blot normalization is required to show that the changes in band intensities correlate to the biological changes in your samples.

Conventional loading controls rely on consistent levels of a reference protein in each sample. Typically, a "housekeeping protein" (HKP) such as β-actin, β-tubulin, or GAPDH is used as a loading control, with the assumption that the expression levels of these proteins remains constant.

The Problem with Housekeeping Proteins

Reliable assessment of the changes in target protein expression levels requires the measurement of both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Unfortunately, housekeeping proteins are usually highly expressed, whereas target proteins are often expressed only in low abundance. Thus, to detect the target protein of interest, large amounts of cell lysate may need to be loaded, resulting in overloading of HKPs, yielding oversaturated reference bands, out of their linear range. Furthermore, HKP expression levels may not be constant but instead vary with different experimental treatments and other factors.

Variation in Housekeeping Protein (HKP) Expression Levels

The expression level of housekeeping proteins can change due to:

  • Experimental conditions
  • Developmental changes
  • Post-transcriptional regulation
  • Differences in cell
  • Tissue age & type

Differences in five candidate housekeeping proteins and total protein staining between tumor and non-cancerous tissues in the validation sample set. Immunodetection measurements of housekeeping protein levels show poor linearity and do not accurately indicate cell lysate loading levels. Total protein normalization of HKPs yields consistent band intensities. Hu X et al. (2016). Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". Oncotarget 7, 66, 679–66, 688.

Total Protein Normalization: The Better Alternative

The use of total protein measurement for western blot loading controls (total protein normalization; TPN) is a method devised to resolve the inherent difficulties with linearity in the immunodetection of both target and control proteins. Total protein levels can be determined by staining the membrane with total protein stains, e.g., Coomassie, SYPRO Ruby, Flamingo, amido black, or Ponceau S.

Because total protein stains are less sensitive than antibody-based immunodetection, they are far less likely to result in an oversaturated signal. As a result, they exhibit good linearity in the common loading range of 10–50 μg of cell lysate. Therefore, both low-abundance target protein levels, measured using sensitive immunodetection techniques, and high-abundance housekeeping protein levels, measured using less sensitive total protein staining techniques, can each be measured in their respective linear dynamic ranges.

Unfortunately, the use of total protein stains can interfere with subsequent blot development and visualization steps, typically requiring tedious staining and destaining procedures, a major reason for the lack of widespread adoption of total protein normalization using these stains. The invention of stain-free imaging technology has made total protein normalization a much better alternative for quantitative western blotting.

Advantages of Total Protein Normalization

Stain-free imaging allows for total protein normalization.

Lane profiling of total protein signal helps remove background and refine band detection.

Total protein normalization takes into account:

  • The intensity of all proteins in the lane
  • Sample loading variations
  • Variations during electrophoresis
  • Variations during transfer

Further Reading: Total Protein Normalization

Stain-Free Imaging

Stain-free imaging technology utilizes a proprietary trihalo compound to enhance natural protein fluorescence by covalently binding to tryptophan residues with a brief UV activation. Images of the gel or membrane after transfer can easily be captured multiple times without staining and destaining steps. This allows visualization, verification, and validation at all steps of electrophoresis and blotting, potentially saving time wasted on western blots with problems that would not otherwise have been detected until the later stages of blot processing and development.

Verification of Western Blotting Protein Transfer Using Stain-Free Imaging

  • Gel before transfer

  • Membrane after transfer

  • Gel after transfer

Assessment of protein transfer using a stain-free enabled imaging system. Images of the gel before and after transfer and of the membrane after transfer were taken using the Criterion Stain-Free imager. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 μg of BSA/lane as a carrier (top band), were electrophoretically separated on a 4–20% 26-well Criterion Stain-Free gel.

Using stain-free total protein measurement as the loading control, researchers can ensure that both the target protein and loading control are measured in the linear dynamic range in a typical western blot experiment. Furthermore, stain-free imaging allows for the complete elimination of the inherently problematic use of housekeeping proteins as loading controls on western blots. Stain-free total protein measurement serves as a more reliable loading control than housekeeping proteins, particularly in the loading range commonly used for cell lysates, permitting the user to obtain truly quantitative western blot data by normalizing bands to total protein in each lane.

Learn More About Stain-Free Technology »

A Better Way to Normalize Quantitative Western Blots

Total protein normalization using stain-free imaging technology allows normalization across a wider dynamic range.

Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10–50 μg of HeLa cell lysate. On the left are representative images of (a), stain-free blot and the chemi blots for (b), β-actin; (c), β-tubulin and (d), GAPDH. Lane labels correspond to total protein load (μg). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted “quantitative response” of actual loading whereas the stain-free signal correlated to the expected result (e). Taylor SC Posch A (2014). The design of a quantitative western blot experiment. Biomed Res Int. 36, 1590

Further Reading: Stain-Free Imaging for Western Blotting

Trends in Protein Separation and Analysis: The Advance of Stain-Free Technology

This article focuses on the theory and concepts surrounding stain-free technology as well as other traditional methods of protein staining.

New Publication Guidelines

Several major scientific journal publishers have recently revised their editorial guidelines for data publication, with an emphasis on reproducibility and quantitation criteria. The Journal of Biological Chemistry guidelines for collecting and presenting data specifically address quantitative western blot publication, providing guidance around normalization methods and supporting data, citing concerns with the use of housekeeping proteins, imaging technology, and antibody specificity.

Bio-Rad's Stain-Free Western Workflow meets the new publication guidelines – does yours?

Meeting the New Publication Requirements for Quantitative Western Blotting

JBC Guidelines: Quantitative Blots How to Meet Requirements Bio-Rad Western Workflow Solutions
"Housekeeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression" Use Total Protein Normalization (TPN) instead of housekeeping proteins The V3 Western Workflow suite simplifies stain-free TPN. This technology is proven with over 100 publications, allows for improved accuracy and will make your data more reliable
"Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range" Use an imaging system with up to 4 logs of dynamic range ChemiDoc Systems are stain-free imagers with the sensitivity of film
Image Lab Software enables simple image generation and quantitation
"A description of the data supporting the specificity of all antibodies is required" Use fully validated antibodies PrecisionAb Antibodies offer documented specificity and validation protocols

Further Reading: Publication Guidelines

  • Nature: Information for Authors & Referees > Policies > Image Integrity and Standards
  • Fosang AJ, Colbran RJ (2015). Transparency Is the Key to Quality. J Biol Chem, 290(50): 29, 692–29, 694
  • McNutt M (2014). Journals Unite for Reproducibility. Science 346(6210): 679
  • Yadav G, Oh K (2018). Defining the New Normal in Quantitative Western Blot Data. Bioradiations.com

Key Factors for Publishing High-Confidence Western Blot Data

Prof. Aldrin V. Gomes (University of California, Davis) relies heavily on biophysical proteomic techniques in his studies of signal transduction, including western blotting. As an editor of several journals, he has routinely observed the submission of low-quality western blot data, leading him and other scientists to question the accuracy of protein quantification by western blotting in prior publications.

Prof. Gomes has deeply examined key success factors in western blotting, including antibody selection and reducing errors and misinterpretations by implementing a standardized procedure and proper controls. Here he presents key factors for high-confidence western blot data:

  • Antibody specificity and blocking reagent quality
  • Properly selected and executed controls
  • Total protein loading control vs. housekeeping protein loading control
     

Prof. Gomes also presents tips to reduce the overall bench time required for first-rate western blots and how to minimize the pain of going through a long procedure only to obtain equivocal results. As the old saying goes, If you didn't have time to do it right the first time, when will you find time to do it again?

The V3 Western Workflow

Bio-Rad’s V3 Western Workflow provides a practical, convenient, and reliable way to perform total protein normalization. Stain-free labeled proteins can be visualized in gels 1 min after gel electrophoresis. Stain-free blot visualization and image acquisition requires only a few seconds using a stain-free enabled imager. This technology makes it easy for researchers to adopt total protein loading control for western blotting.

The V3 Western Workflow System allows you to quickly check electrophoresis and blot transfer quality and obtain truly quantitative western blotting results, updating traditional blotting techniques with innovative tools.

  • Visualize — Stain-free imaging of total proteins in the gel
  • Verify — Confirm transfer, quantify total protein after transfer
  • Validate — Normalize bands to total proteins in Image Lab

Learn More  Request a Demo

 

ChemiDoc MP Imaging System: Imagine No Compromises

With the ChemiDoc MP Imaging System, the superior quantitation of digital imaging meets the sensitivity of film. This best-in-class system images and analyzes gels and western blots. It is designed for multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation, and stain-free technology imaging needs.

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Learn how to properly design a quantitative western blotting experiment and faithfully compare protein expression levels, without concern for changing or overexpressed HKP’s.
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Resources

FEATURED WEBINARUnderstand the Factors That are Crucial to Successful Western Blotting

Successful Western Blotting

Join Bio-Rad western blotting expert Kenneth Oh and his guest speakers for a webinar series that explores the many factors that go into the design and execution of successful and repeatable western blots. Chemiluminescence or fluorescence, qualitative or quantitative blot, not only will we discuss the how-to's, we'll take a deeper dive and discuss the why's.

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  • Reliable Quantification of Western Blot Data

    Dr. S.C Taylor compares results generated for identical blots with camera-based imaging technologies and film. Learn common pitfalls associated with film and densitometric analysis.

  • Designing a Quantitative Western Blot Experiment to Avoid Common Pitfalls

    The quantitative western blot methodology is described with a focus on solid experimental design and data analysis to achieve reproducible, publication quality results in the shortest time frame.

  • Quantitative Western Blotting: Optimization is the Key

    Each step of the quantitative western blot workflow is discussed with a focus on optimization to assure quantitative, reproducible final data that reflect the experimental conditions.

 

Image Lab

  • Image Lab Touch Software Tutorials

    Image Lab Touch Software Resources

    Bio-Rad provides instructional videos for Image Lab Touch Software 2.4, covering the primary applications of the ChemiDoc MP Imaging System. Topics include, logging in, Smart Tray Technology, capturing your image, and exporting data.

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    Image Lab Software Resources

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Application Guides

  • Western Blot Dr. Troubleshooting Guide

    Western Blot Doctor Troubleshooting Guide

    Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.

  • Protein Blotting Guide

    Protein Blotting Guide

    (PDF 7.9 MB)
    Details on blotting technology, available products, and methods, plus tips, techniques, and troubleshooting.

  • Electrophoresis Guide

    Electrophoresis Guide

    (PDF 6.4 MB)
    A guide to polyacrylamide gel electrophoresis and protein detection, including theory, product selection, protocols, and more.

 

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Intel Mac OS X 10_14) AppleWebKit/605.1.15 (KHTML, like Gecko) Version/12.0 Safari/605.1.15] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/total-protein-normalization LSR PODYJQRT8IG9 Total Protein Normalization Total Protein Normalization <div class="pagenav"><img src="/webroot/web/images/opt/prodnav-arrow-top.png" alt="" /> <table border="0"> <tbody> <tr> <td class="pagenavhead"> <h3>On This Page</h3> </td> <td class="pagenavfirst"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Normalization Methods','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#normalization">Normalization Methods</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Stain-Free Imaging','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#stain-free">Stain-Free Imaging</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=New Publication Guidelines','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#publication-guidelines">New Publication Guidelines</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=V3 Western Workflow','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#v3">V3 Western Workflow</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=ChemiDoc Imaging System','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#ChIS">ChemiDoc Imaging System</a></td> <!---<td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Resources','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#webinars">Webinars</a></td>---> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Resources','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#resources">Resources</a></td> <td class="pagenavlast"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Related Products','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#related">Related Products</a></td> </tr> </tbody> </table> </div> <div><a name="normalization"></a></div> <div class="opt-module opt-app-tech-tpn-intro opt-module-noborder no-bttm-pddng"> <h2 class="nextp">Western Blot Normalization Methods&nbsp;</h2> <img src="https://www.bio-rad.com/webroot/web/images/1x1.gif" class="right spacer-img" /> <p>A quantitative western blot accurately measures changes in protein expression by making relative comparisons between different treatments.</p> <p>A properly designed quantitative western blotting experiment ensures that:</p> <ul> <li>The amount of protein loaded is proportional to the signal intensity</li> <li>All quantified targets' signal intensities fall within the linear range of the imaging system</li> <li>Common blot-to-blot variations are accurately assessed by an internal loading control</li> </ul> <p>Western blot normalization allows you to faithfully compare changes in protein expression by establishing the baseline needed to correct against common errors such as inconsistent sample preparation, pipetting error, and uneven protein transfer. Proper western blot normalization is required to show that the changes in band intensities correlate to the biological changes in your samples.</p> <p>Conventional loading controls rely on consistent levels of a reference protein in each sample. Typically, a "housekeeping protein" (HKP) such as &beta;-actin, &beta;-tubulin, or GAPDH is used as a loading control, with the assumption that the expression levels of these proteins remains constant.</p> <h3 class="opt-subhead30">The Problem with Housekeeping Proteins</h3> <p>Reliable assessment of the changes in target protein expression levels requires the measurement of both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Unfortunately, housekeeping proteins are usually highly expressed, whereas target proteins are often expressed only in low abundance. Thus, to detect the target protein of interest, large amounts of cell lysate may need to be loaded, resulting in overloading of HKPs, yielding oversaturated reference bands, out of their linear range. Furthermore, HKP expression levels may not be constant but instead vary with different experimental treatments and other factors.</p> <div class="corner-box topmargin-1em bg-eaf4f6"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/hkp-variations-chart.png" alt="" class="right" /> <h3 class="opt-subhead30">Variation in Housekeeping Protein (HKP) Expression Levels</h3> <p>The expression level of housekeeping proteins can change due to:</p> <ul> <li>Experimental conditions</li> <li>Developmental changes</li> <li>Post-transcriptional regulation</li> <li>Differences in cell</li> <li>Tissue age &amp; type</li> </ul> <p class="caption"><strong>Differences in five candidate housekeeping proteins and total protein staining between tumor and non-cancerous tissues in the validation sample set.</strong> Immunodetection measurements of housekeeping protein levels show poor linearity and do not accurately indicate cell lysate loading levels. Total protein normalization of HKPs yields consistent band intensities. Hu X et al. (2016). Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341829/" target="_blank" rel="noopener noreferrer">Oncotarget</a> 7, 66, 679&ndash;66, 688.</p> </div> <h3 class="opt-subhead30 top-margin">Total Protein Normalization: The Better Alternative</h3> <p>The use of total protein measurement for western blot loading controls (total protein normalization; TPN) is a method devised to resolve the inherent difficulties with linearity in the immunodetection of both target and control proteins. Total protein levels can be determined by staining the membrane with total protein stains, e.g., Coomassie, SYPRO Ruby, Flamingo, amido black, or Ponceau S.</p> <p>Because total protein stains are less sensitive than antibody-based immunodetection, they are far less likely to result in an oversaturated signal. As a result, they exhibit good linearity in the common loading range of 10&ndash;50 &mu;g of cell lysate. Therefore, both low-abundance target protein levels, measured using sensitive immunodetection techniques, and high-abundance housekeeping protein levels, measured using less sensitive total protein staining techniques, can each be measured in their respective linear dynamic ranges.</p> <p>Unfortunately, the use of total protein stains can interfere with subsequent blot development and visualization steps, typically requiring tedious staining and destaining procedures, a major reason for the lack of widespread adoption of total protein normalization using these stains. The invention of stain-free imaging technology has made total protein normalization a much better alternative for quantitative western blotting.</p> <div class="corner-box topmargin-1em bg-edf6e4 corner-box-opt-advantages-tpn"> <h3 class="opt-subhead30">Advantages of Total Protein Normalization</h3> <div class="opt-sub-advantages-tpn right"> <div class="left"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-tpn.png" alt="" /> <p class="caption">Stain-free imaging allows for total protein normalization.</p> </div> <div class="right"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-tpn-profiling.png" alt="" /> <p class="caption">Lane profiling of total protein signal helps remove background and refine band detection.</p> </div> </div> <p>Total protein normalization takes into account:</p> <ul> <li>The intensity of all proteins in the lane</li> <li>Sample loading variations</li> <li>Variations during electrophoresis</li> <li>Variations during transfer</li> </ul> </div> <h3 class="opt-subhead30 top-margin">Further Reading: Total Protein Normalization</h3> <ul class="list-set list-two-col list-set-th"> <li><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/normailizing-western-blots-th.png" alt="" /> <h3><a href="https://www.bioradiations.com/the-how-and-why-of-normalizing-your-western-blots/" target="_blank" rel="noopener noreferrer">The How and Why of Normalizing Your Western Blots</a></h3> </li> <li><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/bulletin-6360-th.png" alt="" /> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_6360.pdf" target="_blank" rel="noopener noreferrer">A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation</a><br /><span class="filesize">(PDF 355 KB)</span></h3> </li> </ul> </div> <div><a name="stain-free"></a></div> <div class="opt-module bg-f0f9f8 opt-module-noborder"> <h2 class="nextp">Stain-Free Imaging</h2> <p>Stain-free imaging technology utilizes a proprietary trihalo compound to enhance natural protein fluorescence by covalently binding to tryptophan residues with a brief UV activation. Images of the gel or membrane after transfer can easily be captured multiple times without staining and destaining steps. This allows visualization, verification, and validation at all steps of electrophoresis and blotting, potentially saving time wasted on western blots with problems that would not otherwise have been detected until the later stages of blot processing and development.</p> <h3 class="opt-subhead30">Verification of Western Blotting Protein Transfer Using Stain-Free Imaging</h3> <ul class="list-set list-three-col"> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/gel-before-transfer.png" alt="" /> <h3>Gel before transfer</h3> </center></li> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/membrane-after-transfer.png" alt="" /> <h3>Membrane after transfer</h3> </center></li> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/gel-after-transfer.png" alt="" /> <h3>Gel after transfer</h3> </center></li> </ul> <p class="caption"><strong>Assessment of protein transfer using a stain-free enabled imaging system.</strong> Images of the gel before and after transfer and of the membrane after transfer were taken using the Criterion Stain-Free imager. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 &mu;g of BSA/lane as a carrier (top band), were electrophoretically separated on a 4&ndash;20% 26-well Criterion Stain-Free gel.</p> <p class="topmargin-1em">Using stain-free total protein measurement as the loading control, researchers can ensure that both the target protein and loading control are measured in the linear dynamic range in a typical western blot experiment. Furthermore, stain-free imaging allows for the complete elimination of the inherently problematic use of housekeeping proteins as loading controls on western blots. Stain-free total protein measurement serves as a more reliable loading control than housekeeping proteins, particularly in the loading range commonly used for cell lysates, permitting the user to obtain truly quantitative western blot data by normalizing bands to total protein in each lane.</p> <p><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Stain-Free&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/stain-free-imaging-technology?ID=NZ0G1815"><strong>Learn More About Stain-Free Technology &raquo;</strong></a></p> <div class="corner-box bg-white topmargin-1em"> <h3 class="opt-subhead30">A Better Way to Normalize Quantitative Western Blots</h3> <p>Total protein normalization using stain-free imaging technology allows normalization across a wider dynamic range.</p> <img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/imaging_and_analysis/technology_detail/acurate-tpn.png" class="topmargin-1em" alt="" style="width: 100%; height: auto;" /> <p class="caption topmargin-1em"><strong>Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10&ndash;50&thinsp;&mu;g of HeLa cell lysate.</strong> On the left are representative images of (a), stain-free blot and the chemi blots for (b), &beta;-actin; (c), &beta;-tubulin and (d), GAPDH. Lane labels correspond to total protein load (&mu;g). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted &ldquo;quantitative response&rdquo; of actual loading whereas the stain-free signal correlated to the expected result (e). Taylor SC Posch A (2014). The design of a quantitative western blot experiment. <a href="https://www.hindawi.com/journals/bmri/2014/361590/" target="_blank" rel="noopener noreferrer">Biomed Res Int.</a> 36, 1590</p> </div> <h3 class="opt-subhead30 top-margin">Further Reading: Stain-Free Imaging for Western Blotting</h3> <img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-western-th.png" alt="" class="left" /> <h3><a href="https://www.bioradiations.com/trends-in-protein-separation-and-analysis-the-advance-of-stain-free-technology/?_ga=2.50353193.1691197357.1557156747-1464263164.1552081756" target="_blank" rel="noopener noreferrer">Trends in Protein Separation and Analysis: The Advance of Stain-Free Technology</a></h3> <p>This article focuses on the theory and concepts surrounding stain-free technology as well as other traditional methods of protein staining.</p> </div> <div><a name="publication-guidelines"></a></div> <div class="opt-module bg-f7f6d3 opt-tpn-pub-guildelines opt-module-noborder"> <h2 class="nextp">New Publication Guidelines</h2> <img src="https://www.bio-rad.com/webroot/web/images/1x1.gif" class="right spacer-img" /> <p>Several major scientific journal publishers have recently revised their editorial guidelines for data publication, with an emphasis on reproducibility and quantitation criteria. The <a href="http://jbcresources.asbmb.org/collecting-and-presenting-data" target="_blank" rel="noopener noreferrer">Journal of Biological Chemistry</a> guidelines for collecting and presenting data specifically address quantitative western blot publication, providing guidance around normalization methods and supporting data, citing concerns with the use of housekeeping proteins, imaging technology, and antibody specificity.</p> <p>Bio-Rad's Stain-Free Western Workflow meets the new publication guidelines &ndash; <span class="forcebreaks">does yours?</span></p> <h3 class="opt-subhead30">Meeting the New Publication Requirements for Quantitative Western Blotting</h3> <table class="opt-module-grid-table opt-module-grid-table-full pd_gridlines-alternate" border="0"> <thead> <tr> <th><strong>JBC Guidelines: Quantitative Blots</strong></th> <th width="33.33%"><strong>How to Meet Requirements</strong></th> <th width="33.33%"><strong>Bio-Rad Western Workflow Solutions</strong></th> </tr> </thead> <tbody> <tr> <td>"Housekeeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression"</td> <td>Use Total Protein Normalization (TPN) instead of housekeeping proteins</td> <td>The <a href="/en-us/product/v3-western-workflow?ID=M08HIB15">V3 Western Workflow</a> suite simplifies stain-free TPN. This technology is proven with over 100 publications, allows for improved accuracy and will make your data more reliable</td> </tr> <tr> <td>"Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range"</td> <td>Use an imaging system with up to 4 logs of dynamic range</td> <td><a href="/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15">ChemiDoc Systems</a> are stain-free imagers with the sensitivity of film<br /> <a href="/en-us/category/image-lab-software-suite?ID=5291f579-0715-48f4-b3de-766b92222582">Image Lab Software</a> enables simple image generation and quantitation</td> </tr> <tr> <td>"A description of the data supporting the specificity of all antibodies is required"</td> <td>Use fully validated antibodies</td> <td><a href="/en-us/product/precisionab-validated-western-blotting-antibodies?ID=N8TPQQ15">PrecisionAb Antibodies</a> offer documented specificity and validation protocols</td> </tr> </tbody> </table> <h3 class="opt-subhead30 top-margin">Further Reading: Publication Guidelines</h3> <ul> <li>Nature: Information for Authors &amp; Referees &gt; Policies &gt; <a href="https://www.nature.com/authors/policies/image.html" target="_blank" rel="noopener noreferrer">Image Integrity and Standards</a></li> <li>Fosang AJ, Colbran RJ (2015). Transparency Is the Key to Quality. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705984/" target="_blank" rel="noopener noreferrer">J Biol Chem</a>, 290(50): 29, 692&ndash;29, 694</li> <li>McNutt M (2014). Journals Unite for Reproducibility. <a href="https://science.sciencemag.org/content/346/6210/679.full" target="_blank" rel="noopener noreferrer">Science</a> 346(6210): 679</li> <li>Yadav G, Oh K (2018). Defining the New Normal in Quantitative Western Blot Data. <a href="https://www.bioradiations.com/defining-the-new-normal-in-quantitative-western-blot-data/" target="_blank" rel="noopener noreferrer">Bioradiations.com</a></li> </ul> <h3 class="opt-subhead30">Key Factors for Publishing High-Confidence Western Blot Data</h3> <p>Prof. Aldrin V. Gomes (University of California, Davis) relies heavily on biophysical proteomic techniques in his studies of signal transduction, including western blotting. As an editor of several journals, he has routinely observed the submission of low-quality western blot data, leading him and other scientists to question the accuracy of protein quantification by western blotting in prior publications.</p> <iframe src="https://www.youtube.com/embed/zrfAHAo4xT0?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" _fsrb="true" class="right"></iframe> <p>Prof. Gomes has deeply examined key success factors in western blotting, including antibody selection and reducing errors and misinterpretations by implementing a standardized procedure and proper controls. Here he presents key factors for high-confidence western blot data:</p> <ul> <li>Antibody specificity and blocking reagent quality</li> <li>Properly selected and executed controls</li> <li>Total protein loading control vs. housekeeping protein loading control<br />&nbsp;</li> </ul> <p>Prof. Gomes also presents tips to reduce the overall bench time required for first-rate western blots and how to minimize the pain of going through a long procedure only to obtain equivocal results. As the old saying goes, If you didn't have time to do it right the first time, when will you find time to do it again?</p> </div> <div><a name="v3"></a></div> <div class="opt-module opt-module-noborder"> <h2 class="nextp">The V3 Western Workflow</h2> <p>Bio-Rad&rsquo;s V3 Western Workflow provides a practical, convenient, and reliable way to perform total protein normalization. Stain-free labeled proteins can be visualized in gels 1 min after gel electrophoresis. Stain-free blot visualization and image acquisition requires only a few seconds using a stain-free enabled imager. This technology makes it easy for researchers to adopt total protein loading control for western blotting.</p> <iframe src="https://www.youtube.com/embed/RWsDtTqDW_o?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" class="right"></iframe> <p>The V3 Western Workflow System allows you to quickly check electrophoresis and blot transfer quality and obtain truly quantitative western blotting results, updating traditional blotting techniques with innovative tools.</p> <ul> <li><strong>Visualize</strong> &mdash; Stain-free imaging of total proteins in the gel</li> <li><strong>Verify</strong> &mdash; Confirm transfer, quantify total protein after transfer</li> <li><strong>Validate</strong> &mdash; Normalize bands to total proteins in Image Lab</li> </ul> <p><a class="bttn-block linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=V3&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/product/v3-western-workflow"><span class="bttn-orange">Learn More</span></a> &nbsp;<a class="bttn-block no-target-style" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=V3&amp;tgp_pcp_link=Request a Demo','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="https://info.bio-rad.com/ww-V3-Central-Umbrella-btwas.html" target="_blank" rel="noopener noreferrer"><span class="bttn-green">Request a Demo</span></a></p> <div class="dotteddivider topmargin-1em">&nbsp;</div> <iframe src="https://www.youtube.com/embed/xAUFWRONV_U?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" mce_src="https://www.youtube.com/embed/nVsLxeT8Reg?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" _fsrb="true" class="left"></iframe> <h3 class="opt-subhead">ChemiDoc MP Imaging System: Imagine No Compromises</h3> <p>With the ChemiDoc MP Imaging System, the superior quantitation of digital imaging meets the sensitivity of film. This best-in-class system images and analyzes gels and western blots. It is designed for multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation, and stain-free technology imaging needs.</p> <p><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=ChemiDocMP&amp;tgp_pcp_link=Learn More','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/product/chemidoc-mp-imaging-system?ID=NINJ8ZE8Z"><strong>Learn More &raquo;</strong></a></p> </div> <div><a name="ChIS"></a></div> <div class="opt-module opt-module-noborder bg-f0f9f8"> <h2>ChemiDoc Imaging Systems</h2> <h3 class="opt-subhead30 top-margin-neg">Complete Capabilities: Superior Images</h3> <div id="interactiveRibbonOuter"> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/stain-free-square.png'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Stain-Free<br /><small>Total Protein Normalization</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Learn how to properly design a quantitative western blotting experiment and faithfully compare protein expression levels, without concern for changing or overexpressed HKP&rsquo;s. <br /><a href="/en-sg/applications-technologies/total-protein-normalization?ID=PODYJQRT8IG9">See Data &raquo;</a></p> </div> </div> </div> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/chemiluminescence.jpg'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Chemiluminescence<br /><small>Replaces Film</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Save money on consumables and reduce environmental waste without compromising on performance. Get the sensitivity of film and the convenience of digital documentation. <br /><a href="/en-sg/applications-technologies/comparing-x-ray-film-digital-imaging-systems?ID=PNK1ECTU86LJ">See Data &raquo;</a></p> </div> </div> </div> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/bio-rad-fluorescence.jpg'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Multiplex Fluorescence<br /><small>3 Colors</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Save sample, avoid the errors commonly associated with stripping, reprobing, and cutting blots and answer more complex biological questions by optimizing your experiments to fluorescence. <br /><a href="/en-sg/applications-technologies/fluorescent-western-blotting?ID=PJW4ANE08O1Y">See Data &raquo;</a></p> </div> </div> </div> </div> </div> <div class="opt-module opt-module-precisionab-feature opt-module-noborder linkpointer hidden US-only AU-only NZ-only SG-only"> <h2 class="nextp white"><span class="featuredtxt">FEATURED PRODUCT</span>PrecisionAb Validated Primary Antibodies<br />for Western Blotting</h2> <ul class="list-orange-arrow white"> <li>Comprehensive selection of antibodies for cancer and neuroscience research</li> <li>Highly specific and sensitive</li> <li>Validated against up to 12 lysates</li> <li>Stringent QC criteria for superior consistency</li> </ul> <p><a class="bttn-block" onmousedown="dcsMultiTrack('DCS.dcsqry','? tgp_pcp_link_group=PrecisionAb Validated Western Blotting Antibodies&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999'); return false;" href="/en-us/product/precisionab-validated-western-blotting-antibodies"><span class="bttn-orange">Find Your Antibody</span></a></p> </div> <!---<div><a name="webinars"></a></div> <div class="opt-module opt-module-noborder"> <h2 class="nextp">Webinars</h2> </div>---> <div><a name="resources"></a></div> <div class="opt-module optresources-bg"> <h2>Resources</h2> <div class="corner-box opt-western-blot-webinar"> <h3 class="opt-subhead30 white"><span class="featuredtxt">FEATURED WEBINAR</span>Understand the Factors That are Crucial to Successful Western Blotting</h3> <img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/kenneth-oh-th.jpg" alt="Successful Western Blotting" class="left" /> <p>Join Bio-Rad western blotting expert Kenneth Oh and his guest speakers for a webinar series that explores the many factors that go into the design and execution of successful and repeatable western blots. Chemiluminescence or fluorescence, qualitative or quantitative blot, not only will we discuss the how-to's, we'll take a deeper dive and discuss the why's.<br /><br /><a class="bttn-block no-target-style" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Featured Webinar&amp;tgp_pcp_link=View Webinar','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="https://info.bio-rad.com/ww-PQDwebinars.html" target="_blank" rel="noopener noreferrer"><span class="bttn-orange">View Webinar</span></a></p> </div> <br /> <ul class="list-set list-three-col"> <li><iframe src="https://www.youtube.com/embed/q0T_Af7khHw?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Reliable Quantification of Western Blot Data</h3> <p>Dr. S.C Taylor compares results generated for identical blots with camera-based imaging technologies and film. Learn common pitfalls associated with film and densitometric analysis.</p> </li> <li><iframe src="https://www.youtube.com/embed/iCgmeufGM2g?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Designing a Quantitative Western Blot Experiment to Avoid Common Pitfalls</h3> <p>The quantitative western blot methodology is described with a focus on solid experimental design and data analysis to achieve reproducible, publication quality results in the shortest time frame.</p> </li> <li><iframe src="https://www.youtube.com/embed/QXg0r_DVlAs?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Quantitative Western Blotting: Optimization is the Key</h3> <p>Each step of the quantitative western blot workflow is discussed with a focus on optimization to assure quantitative, reproducible final data that reflect the experimental conditions.</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Image Lab</h3> <ul class="list-set list-two-col list-set-th"> <li><a href="/en-us/category/image-lab-touch-resources?ID=PJWAQ6E08O1Y "><img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/image-lab-touch-video-th.gif" alt="Image Lab Touch Software Tutorials" /></a> <h3><a href="/en-us/category/image-lab-touch-resources?ID=PJWAQ6E08O1Y ">Image Lab Touch Software Resources</a></h3> <p>Bio-Rad provides instructional videos for Image Lab Touch Software 2.4, covering the primary applications of the ChemiDoc MP Imaging System. Topics include, logging in, Smart Tray Technology, capturing your image, and exporting data.</p> </li> <li><a href="/en-us/category/image-lab-software-resources?ID=PJWA0VTU86LJ"><img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/image-lab-video-th.gif" alt="Image Lab Software Tutorials" /></a> <h3><a href="/en-us/category/image-lab-software-resources?ID=PJWA0VTU86LJ">Image Lab Software Resources</a></h3> <p>Bio-Rad provides tutorials for basic acquisition and advanced analysis use of Image Lab Software 6.0.1. Topics include, densitometric analysis, molecular weight, normalization, purity assessment, and more!</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Application Guides</h3> <ul class="list-set list-three-col"> <li><a class="linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Western Blot Dr Troubleshooting Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-dr-troubleshooting.jpg" alt="Western Blot Dr. Troubleshooting Guide" /></a> <h3><a class="linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Western Blot Dr Troubleshooting Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor">Western Blot Doctor Troubleshooting Guide</a></h3> <p>Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look <span class="forcebreaks">their best.</span></p> </li> <li><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Protein Blotting Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-blotting-guide.jpg" alt="Protein Blotting Guide" /></a> <h3><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Protein Blotting Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Protein Blotting Guide</a></h3> <p><span class="filesize">(PDF 7.9 MB)</span><br />Details on blotting technology, available products, and methods, plus tips, techniques, and troubleshooting.</p> </li> <li><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Electrophoresis Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-electrophoresis-guide.jpg" alt="Electrophoresis Guide" /></a> <h3><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Electrophoresis Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Electrophoresis Guide</a></h3> <p><span class="filesize">(PDF 6.4 MB)</span><br />A guide to polyacrylamide gel electrophoresis and protein detection, including theory, product selection, protocols, and more.</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Documents Library</h3> <ul class="list-set list-two-col list-set-th"> <li><a href="/webroot/web/pdf/lsr/literature/Bulletin_6873.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/MA17-0012_S3_CV1_T2_01.png" /></a> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_6873.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">ChemiDoc MP System Brochure</a><br /><span class="filesize">(PDF 1.26 MB)</span></h3> <p>Explore the features and benefits of the new high-end imaging system for the best fluorescence and chemiluminescence detection.</p> </li> <li><a href="/webroot/web/pdf/lsr/literature/Bulletin_RP0051.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/MA17-0012_S3_CV1_T1_04.png" /> </a> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_RP0051.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Stain-Free Approach to Western Blotting</a><br /><span class="filesize">(PDF 864 KB)</span></h3> <p>Learn how to use stain-free technology for total protein normalization as an alternative to the standard blot normalization process.</p> </li> <li><a 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This improves precision, cost-effectiveness, and data reliability. total protein normalization, TPN, western blot loading controls, immunodetection target proteins, immunodetection control proteins 05/07/19 06:33 PM 05/07/29 08:39 AM AR,BO,BR,CA,CL,CO,CR,DO,EC,SV,GU,GT,HT,HN,JM,MX,NI,PA,PY,PE,PR,TT,US,UY,VE,AU,BD,BN,KH,CN,HK,IN,ID,JP,KR,MO,MY,FM,NP,NZ,PW,PG,PH,SG,SB,LK,TW,TH,TO,VU,VN,AL,DZ,AI,AM,AT,AZ,BH,BE,BA,BW,BG,BF,CM,HR,CY,CZ,DK,EG,ER,EE,ET,FO,FI,FR,GF,PF,GA,GE,DE,GH,GR,GP,HU,IS,IE,IL,IT,JO,KZ,KE,XK,KW,LV,LB,LI,LT,LU,MK,MG,ML,MT,MQ,MU,MD,MS,MA,NL,NG,NO,OM,PK,PS,PL,PT,QA,RO,RU,ST,SA,SN,RS,SK,SI,ZA,ES,SE,CH,TZ,TG,TN,TR,UG,UA,AE,UK,UZ,VA,EH,YE LSR /LSR/Technologies/Western_Blotting N 0 <div class="pagenav"><img src="/webroot/web/images/opt/prodnav-arrow-top.png" alt="" /> <table border="0"> <tbody> <tr> <td class="pagenavhead"> <h3>On This Page</h3> </td> <td class="pagenavfirst"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Normalization Methods','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#normalization">Normalization Methods</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Stain-Free Imaging','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#stain-free">Stain-Free Imaging</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=New Publication Guidelines','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#publication-guidelines">New Publication Guidelines</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=V3 Western Workflow','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#v3">V3 Western Workflow</a></td> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=ChemiDoc Imaging System','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#ChIS">ChemiDoc Imaging System</a></td> <!---<td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Resources','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#webinars">Webinars</a></td>---> <td><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Resources','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#resources">Resources</a></td> <td class="pagenavlast"><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Page Nav&amp;tgp_pcp_link=Related Products','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="#related">Related Products</a></td> </tr> </tbody> </table> </div> <div><a name="normalization"></a></div> <div class="opt-module opt-app-tech-tpn-intro opt-module-noborder no-bttm-pddng"> <h2 class="nextp">Western Blot Normalization Methods</h2> <img src="https://www.bio-rad.com/webroot/web/images/1x1.gif" class="right spacer-img" /> <p>A quantitative western blot accurately measures changes in protein expression by making relative comparisons between different treatments.</p> <p>A properly designed quantitative western blotting experiment ensures that:</p> <ul> <li>The amount of protein loaded is proportional to the signal intensity</li> <li>All quantified targets' signal intensities fall within the linear range of the imaging system</li> <li>Common blot-to-blot variations are accurately assessed by an internal loading control</li> </ul> <p>Western blot normalization allows you to faithfully compare changes in protein expression by establishing the baseline needed to correct against common errors such as inconsistent sample preparation, pipetting error, and uneven protein transfer. Proper western blot normalization is required to show that the changes in band intensities correlate to the biological changes in your samples.</p> <p>Conventional loading controls rely on consistent levels of a reference protein in each sample. Typically, a "housekeeping protein" (HKP) such as &beta;-actin, &beta;-tubulin, or GAPDH is used as a loading control, with the assumption that the expression levels of these proteins remains constant.</p> <h3 class="opt-subhead30">The Problem with Housekeeping Proteins</h3> <p>Reliable assessment of the changes in target protein expression levels requires the measurement of both the target protein and the loading control protein in their linear dynamic ranges for immunodetection. Unfortunately, housekeeping proteins are usually highly expressed, whereas target proteins are often expressed only in low abundance. Thus, to detect the target protein of interest, large amounts of cell lysate may need to be loaded, resulting in overloading of HKPs, yielding oversaturated reference bands, out of their linear range. Furthermore, HKP expression levels may not be constant but instead vary with different experimental treatments and other factors.</p> <div class="corner-box topmargin-1em bg-eaf4f6"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/hkp-variations-chart.png" alt="" class="right" /> <h3 class="opt-subhead30">Variation in Housekeeping Protein (HKP) Expression Levels</h3> <p>The expression level of housekeeping proteins can change due to:</p> <ul> <li>Experimental conditions</li> <li>Developmental changes</li> <li>Post-transcriptional regulation</li> <li>Differences in cell</li> <li>Tissue age &amp; type</li> </ul> <p class="caption"><strong>Differences in five candidate housekeeping proteins and total protein staining between tumor and non-cancerous tissues in the validation sample set.</strong> Immunodetection measurements of housekeeping protein levels show poor linearity and do not accurately indicate cell lysate loading levels. Total protein normalization of HKPs yields consistent band intensities. Hu X et al. (2016). Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341829/" target="_blank" rel="noopener noreferrer">Oncotarget</a> 7, 66, 679&ndash;66, 688.</p> </div> <h3 class="opt-subhead30 top-margin">Total Protein Normalization: The Better Alternative</h3> <p>The use of total protein measurement for western blot loading controls (total protein normalization; TPN) is a method devised to resolve the inherent difficulties with linearity in the immunodetection of both target and control proteins. Total protein levels can be determined by staining the membrane with total protein stains, e.g., Coomassie, SYPRO Ruby, Flamingo, amido black, or Ponceau S.</p> <p>Because total protein stains are less sensitive than antibody-based immunodetection, they are far less likely to result in an oversaturated signal. As a result, they exhibit good linearity in the common loading range of 10&ndash;50 &mu;g of cell lysate. Therefore, both low-abundance target protein levels, measured using sensitive immunodetection techniques, and high-abundance housekeeping protein levels, measured using less sensitive total protein staining techniques, can each be measured in their respective linear dynamic ranges.</p> <p>Unfortunately, the use of total protein stains can interfere with subsequent blot development and visualization steps, typically requiring tedious staining and destaining procedures, a major reason for the lack of widespread adoption of total protein normalization using these stains. The invention of stain-free imaging technology has made total protein normalization a much better alternative for quantitative western blotting.</p> <div class="corner-box topmargin-1em bg-edf6e4 corner-box-opt-advantages-tpn"> <h3 class="opt-subhead30">Advantages of Total Protein Normalization</h3> <div class="opt-sub-advantages-tpn right"> <div class="left"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-tpn.png" alt="" /> <p class="caption">Stain-free imaging allows for total protein normalization.</p> </div> <div class="right"><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-tpn-profiling.png" alt="" /> <p class="caption">Lane profiling of total protein signal helps remove background and refine band detection.</p> </div> </div> <p>Total protein normalization takes into account:</p> <ul> <li>The intensity of all proteins in the lane</li> <li>Sample loading variations</li> <li>Variations during electrophoresis</li> <li>Variations during transfer</li> </ul> </div> <h3 class="opt-subhead30 top-margin">Further Reading: Total Protein Normalization</h3> <ul class="list-set list-two-col list-set-th"> <li><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/normailizing-western-blots-th.png" alt="" /> <h3><a href="https://www.bioradiations.com/the-how-and-why-of-normalizing-your-western-blots/" target="_blank" rel="noopener noreferrer">The How and Why of Normalizing Your Western Blots</a></h3> </li> <li><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/bulletin-6360-th.png" alt="" /> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_6360.pdf" target="_blank" rel="noopener noreferrer">A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation</a><br /><span class="filesize">(PDF 355 KB)</span></h3> </li> </ul> </div> <div><a name="stain-free"></a></div> <div class="opt-module bg-f0f9f8 opt-module-noborder"> <h2 class="nextp">Stain-Free Imaging</h2> <p>Stain-free imaging technology utilizes a proprietary trihalo compound to enhance natural protein fluorescence by covalently binding to tryptophan residues with a brief UV activation. Images of the gel or membrane after transfer can easily be captured multiple times without staining and destaining steps. This allows visualization, verification, and validation at all steps of electrophoresis and blotting, potentially saving time wasted on western blots with problems that would not otherwise have been detected until the later stages of blot processing and development.</p> <h3 class="opt-subhead30">Verification of Western Blotting Protein Transfer Using Stain-Free Imaging</h3> <ul class="list-set list-three-col"> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/gel-before-transfer.png" alt="" /> <h3>Gel before transfer</h3> </center></li> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/membrane-after-transfer.png" alt="" /> <h3>Membrane after transfer</h3> </center></li> <li><center><img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/gel-after-transfer.png" alt="" /> <h3>Gel after transfer</h3> </center></li> </ul> <p class="caption"><strong>Assessment of protein transfer using a stain-free enabled imaging system.</strong> Images of the gel before and after transfer and of the membrane after transfer were taken using the Criterion Stain-Free imager. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 &mu;g of BSA/lane as a carrier (top band), were electrophoretically separated on a 4&ndash;20% 26-well Criterion Stain-Free gel.</p> <p class="topmargin-1em">Using stain-free total protein measurement as the loading control, researchers can ensure that both the target protein and loading control are measured in the linear dynamic range in a typical western blot experiment. Furthermore, stain-free imaging allows for the complete elimination of the inherently problematic use of housekeeping proteins as loading controls on western blots. Stain-free total protein measurement serves as a more reliable loading control than housekeeping proteins, particularly in the loading range commonly used for cell lysates, permitting the user to obtain truly quantitative western blot data by normalizing bands to total protein in each lane.</p> <p><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Stain-Free&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/stain-free-imaging-technology?ID=NZ0G1815"><strong>Learn More About Stain-Free Technology &raquo;</strong></a></p> <div class="corner-box bg-white topmargin-1em"> <h3 class="opt-subhead30">A Better Way to Normalize Quantitative Western Blots</h3> <p>Total protein normalization using stain-free imaging technology allows normalization across a wider dynamic range.</p> <img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/imaging_and_analysis/technology_detail/acurate-tpn.png" class="topmargin-1em" alt="" style="width: 100%; height: auto;" /> <p class="caption topmargin-1em"><strong>Linearity comparison of stain-free total protein measurement and immunodetection of three housekeeping proteins in 10&ndash;50&thinsp;&mu;g of HeLa cell lysate.</strong> On the left are representative images of (a), stain-free blot and the chemi blots for (b), &beta;-actin; (c), &beta;-tubulin and (d), GAPDH. Lane labels correspond to total protein load (&mu;g). Although the actin and tubulin signals appear linear, the densitometric ratio was far below the predicted &ldquo;quantitative response&rdquo; of actual loading whereas the stain-free signal correlated to the expected result (e). Taylor SC Posch A (2014). The design of a quantitative western blot experiment. <a href="https://www.hindawi.com/journals/bmri/2014/361590/" target="_blank" rel="noopener noreferrer">Biomed Res Int.</a> 36, 1590</p> </div> <h3 class="opt-subhead30 top-margin">Further Reading: Stain-Free Imaging for Western Blotting</h3> <img src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/stain-free-western-th.png" alt="" class="left" /> <h3><a href="https://www.bioradiations.com/trends-in-protein-separation-and-analysis-the-advance-of-stain-free-technology/?_ga=2.50353193.1691197357.1557156747-1464263164.1552081756" target="_blank" rel="noopener noreferrer">Trends in Protein Separation and Analysis: The Advance of Stain-Free Technology</a></h3> <p>This article focuses on the theory and concepts surrounding stain-free technology as well as other traditional methods of protein staining.</p> </div> <div><a name="publication-guidelines"></a></div> <div class="opt-module bg-f7f6d3 opt-tpn-pub-guildelines opt-module-noborder"> <h2 class="nextp">New Publication Guidelines</h2> <img src="https://www.bio-rad.com/webroot/web/images/1x1.gif" class="right spacer-img" /> <p>Several major scientific journal publishers have recently revised their editorial guidelines for data publication, with an emphasis on reproducibility and quantitation criteria. The <a href="http://jbcresources.asbmb.org/collecting-and-presenting-data" target="_blank" rel="noopener noreferrer">Journal of Biological Chemistry</a> guidelines for collecting and presenting data specifically address quantitative western blot publication, providing guidance around normalization methods and supporting data, citing concerns with the use of housekeeping proteins, imaging technology, and antibody specificity.</p> <p>Bio-Rad's Stain-Free Western Workflow meets the new publication guidelines &ndash; <span class="forcebreaks">does yours?</span></p> <h3 class="opt-subhead30">Meeting the New Publication Requirements for Quantitative Western Blotting</h3> <table class="opt-module-grid-table opt-module-grid-table-full pd_gridlines-alternate" border="0"> <thead> <tr> <th><strong>JBC Guidelines: Quantitative Blots</strong></th> <th width="33.33%"><strong>How to Meet Requirements</strong></th> <th width="33.33%"><strong>Bio-Rad Western Workflow Solutions</strong></th> </tr> </thead> <tbody> <tr> <td>"Housekeeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression"</td> <td>Use Total Protein Normalization (TPN) instead of housekeeping proteins</td> <td>The <a href="/en-us/product/v3-western-workflow?ID=M08HIB15">V3 Western Workflow</a> suite simplifies stain-free TPN. This technology is proven with over 100 publications, allows for improved accuracy and will make your data more reliable</td> </tr> <tr> <td>"Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range"</td> <td>Use an imaging system with up to 4 logs of dynamic range</td> <td><a href="/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15">ChemiDoc Systems</a> are stain-free imagers with the sensitivity of film<br /> <a href="/en-us/category/image-lab-software-suite?ID=5291f579-0715-48f4-b3de-766b92222582">Image Lab Software</a> enables simple image generation and quantitation</td> </tr> <tr> <td>"A description of the data supporting the specificity of all antibodies is required"</td> <td>Use fully validated antibodies</td> <td><a href="/en-us/product/precisionab-validated-western-blotting-antibodies?ID=N8TPQQ15">PrecisionAb Antibodies</a> offer documented specificity and validation protocols</td> </tr> </tbody> </table> <h3 class="opt-subhead30 top-margin">Further Reading: Publication Guidelines</h3> <ul> <li>Nature: Information for Authors &amp; Referees &gt; Policies &gt; <a href="https://www.nature.com/authors/policies/image.html" target="_blank" rel="noopener noreferrer">Image Integrity and Standards</a></li> <li>Fosang AJ, Colbran RJ (2015). Transparency Is the Key to Quality. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705984/" target="_blank" rel="noopener noreferrer">J Biol Chem</a>, 290(50): 29, 692&ndash;29, 694</li> <li>McNutt M (2014). Journals Unite for Reproducibility. <a href="https://science.sciencemag.org/content/346/6210/679.full" target="_blank" rel="noopener noreferrer">Science</a> 346(6210): 679</li> <li>Yadav G, Oh K (2018). Defining the New Normal in Quantitative Western Blot Data. <a href="https://www.bioradiations.com/defining-the-new-normal-in-quantitative-western-blot-data/" target="_blank" rel="noopener noreferrer">Bioradiations.com</a></li> </ul> <h3 class="opt-subhead30">Key Factors for Publishing High-Confidence Western Blot Data</h3> <p>Prof. Aldrin V. Gomes (University of California, Davis) relies heavily on biophysical proteomic techniques in his studies of signal transduction, including western blotting. As an editor of several journals, he has routinely observed the submission of low-quality western blot data, leading him and other scientists to question the accuracy of protein quantification by western blotting in prior publications.</p> <iframe src="https://www.youtube.com/embed/zrfAHAo4xT0?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" _fsrb="true" class="right"></iframe> <p>Prof. Gomes has deeply examined key success factors in western blotting, including antibody selection and reducing errors and misinterpretations by implementing a standardized procedure and proper controls. Here he presents key factors for high-confidence western blot data:</p> <ul> <li>Antibody specificity and blocking reagent quality</li> <li>Properly selected and executed controls</li> <li>Total protein loading control vs. housekeeping protein loading control<br />&nbsp;</li> </ul> <p>Prof. Gomes also presents tips to reduce the overall bench time required for first-rate western blots and how to minimize the pain of going through a long procedure only to obtain equivocal results. As the old saying goes, If you didn't have time to do it right the first time, when will you find time to do it again?</p> </div> <div><a name="v3"></a></div> <div class="opt-module opt-module-noborder"> <h2 class="nextp">The V3 Western Workflow</h2> <p>Bio-Rad&rsquo;s V3 Western Workflow provides a practical, convenient, and reliable way to perform total protein normalization. Stain-free labeled proteins can be visualized in gels 1 min after gel electrophoresis. Stain-free blot visualization and image acquisition requires only a few seconds using a stain-free enabled imager. This technology makes it easy for researchers to adopt total protein loading control for western blotting.</p> <iframe src="https://www.youtube.com/embed/RWsDtTqDW_o?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" class="right"></iframe> <p>The V3 Western Workflow System allows you to quickly check electrophoresis and blot transfer quality and obtain truly quantitative western blotting results, updating traditional blotting techniques with innovative tools.</p> <ul> <li><strong>Visualize</strong> &mdash; Stain-free imaging of total proteins in the gel</li> <li><strong>Verify</strong> &mdash; Confirm transfer, quantify total protein after transfer</li> <li><strong>Validate</strong> &mdash; Normalize bands to total proteins in Image Lab</li> </ul> <p><a class="bttn-block linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=V3&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/product/v3-western-workflow"><span class="bttn-orange">Learn More</span></a> &nbsp;<a class="bttn-block no-target-style" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=V3&amp;tgp_pcp_link=Request a Demo','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="https://info.bio-rad.com/ww-V3-Central-Umbrella-btwas.html" target="_blank" rel="noopener noreferrer"><span class="bttn-green">Request a Demo</span></a></p> <div class="dotteddivider topmargin-1em">&nbsp;</div> <iframe src="https://www.youtube.com/embed/xAUFWRONV_U?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" mce_src="https://www.youtube.com/embed/nVsLxeT8Reg?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="245px" width="437px" _fsrb="true" class="left"></iframe> <h3 class="opt-subhead">ChemiDoc MP Imaging System: Imagine No Compromises</h3> <p>With the ChemiDoc MP Imaging System, the superior quantitation of digital imaging meets the sensitivity of film. This best-in-class system images and analyzes gels and western blots. It is designed for multiplex fluorescent western blotting, chemiluminescence detection, general gel documentation, and stain-free technology imaging needs.</p> <p><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=ChemiDocMP&amp;tgp_pcp_link=Learn More','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/product/chemidoc-mp-imaging-system?ID=NINJ8ZE8Z"><strong>Learn More &raquo;</strong></a></p> </div> <div><a name="ChIS"></a></div> <div class="opt-module opt-module-noborder bg-f9f9e4"> <h2>ChemiDoc Imaging System</h2> <h3 class="opt-subhead30 top-margin-neg">Better Options. Better Results.</h3> <div id="interactiveRibbonOuter"> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/stain-free-square.png'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Stain-Free<br /><small>Total Protein Normalization</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Learn how to properly design a quantitative western blotting experiment and faithfully compare protein expression levels, without concern for changing or overexpressed HKP&rsquo;s. <br /><a href="/en-sg/applications-technologies/total-protein-normalization?ID=PODYJQRT8IG9">See Data &raquo;</a></p> </div> </div> </div> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/chemiluminescence.jpg'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Chemiluminescence<br /><small>Replaces Film</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Save money on consumables and reduce environmental waste without compromising on performance. Get the sensitivity of film and the convenience of digital documentation. <br /><a href="/en-sg/applications-technologies/comparing-x-ray-film-digital-imaging-systems?ID=PNK1ECTU86LJ">See Data &raquo;</a></p> </div> </div> </div> <div class="ribbonThird" style="background-image: url('/webroot/web/images/lsr/global/english/solutions/technologies/Fluorescent-Western-Blotting/bio-rad-fluorescence.jpg'); background-repeat: no-repeat;"> <div class="ribbonThirdInner"> <div class="ribbonHeader"> <div class="ribbon2"> <div class="ribbon2Center"> <h4 class="gy05">Multiplex Fluorescence<br /><small>3 Colors</small></h4> </div> </div> <div class="ribbon2Right"></div> </div> <div class="ribbonDesc"> <p>Save sample, avoid the errors commonly associated with stripping, reprobing, and cutting blots and answer more complex biological questions by optimizing your experiments to fluorescence. <br /><a href="/en-sg/applications-technologies/fluorescent-western-blotting?ID=PJW4ANE08O1Y">See Data &raquo;</a></p> </div> </div> </div> </div> </div> <div class="opt-module opt-module-precisionab-feature opt-module-noborder linkpointer hidden US-only AU-only NZ-only SG-only"> <h2 class="nextp white"><span class="featuredtxt">FEATURED PRODUCT</span>PrecisionAb Validated Primary Antibodies<br />for Western Blotting</h2> <ul class="list-orange-arrow white"> <li>Comprehensive selection of antibodies for cancer and neuroscience research</li> <li>Highly specific and sensitive</li> <li>Validated against up to 12 lysates</li> <li>Stringent QC criteria for superior consistency</li> </ul> <p><a class="bttn-block" onmousedown="dcsMultiTrack('DCS.dcsqry','? tgp_pcp_link_group=PrecisionAb Validated Western Blotting Antibodies&amp;tgp_pcp_link=Learn more','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999'); return false;" href="/en-us/product/precisionab-validated-western-blotting-antibodies"><span class="bttn-orange">Find Your Antibody</span></a></p> </div> <!---<div><a name="webinars"></a></div> <div class="opt-module opt-module-noborder"> <h2 class="nextp">Webinars</h2> </div>---> <div><a name="resources"></a></div> <div class="opt-module optresources-bg"> <h2>Resources</h2> <div class="corner-box opt-western-blot-webinar"> <h3 class="opt-subhead30 white"><span class="featuredtxt">FEATURED WEBINAR</span>Understand the Factors That are Crucial to Successful Western Blotting</h3> <img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/kenneth-oh-th.jpg" alt="Successful Western Blotting" class="left" /> <p>Join Bio-Rad western blotting expert Kenneth Oh and his guest speakers for a webinar series that explores the many factors that go into the design and execution of successful and repeatable western blots. Chemiluminescence or fluorescence, qualitative or quantitative blot, not only will we discuss the how-to's, we'll take a deeper dive and discuss the why's.<br /><br /><a class="bttn-block no-target-style" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Featured Webinar&amp;tgp_pcp_link=View Webinar','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="https://info.bio-rad.com/ww-PQDwebinars.html" target="_blank" rel="noopener noreferrer"><span class="bttn-orange">View Webinar</span></a></p> </div> <br /> <ul class="list-set list-three-col"> <li><iframe src="https://www.youtube.com/embed/q0T_Af7khHw?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Reliable Quantification of Western Blot Data</h3> <p>Dr. S.C Taylor compares results generated for identical blots with camera-based imaging technologies and film. Learn common pitfalls associated with film and densitometric analysis.</p> </li> <li><iframe src="https://www.youtube.com/embed/iCgmeufGM2g?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Designing a Quantitative Western Blot Experiment to Avoid Common Pitfalls</h3> <p>The quantitative western blot methodology is described with a focus on solid experimental design and data analysis to achieve reproducible, publication quality results in the shortest time frame.</p> </li> <li><iframe src="https://www.youtube.com/embed/QXg0r_DVlAs?version=3&amp;rel=0&amp;showinfo=0&amp;theme=light&amp;modestbranding=1&amp;fs=1;wmode=transparent" height="151px" width="270px" _fsrb="true"></iframe> <h3>Quantitative Western Blotting: Optimization is the Key</h3> <p>Each step of the quantitative western blot workflow is discussed with a focus on optimization to assure quantitative, reproducible final data that reflect the experimental conditions.</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Image Lab</h3> <ul class="list-set list-two-col list-set-th"> <li><a href="/en-us/category/image-lab-touch-resources?ID=PJWAQ6E08O1Y "><img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/image-lab-touch-video-th.gif" alt="Image Lab Touch Software Tutorials" /></a> <h3><a href="/en-us/category/image-lab-touch-resources?ID=PJWAQ6E08O1Y ">Image Lab Touch Software Resources</a></h3> <p>Bio-Rad provides instructional videos for Image Lab Touch Software 2.4, covering the primary applications of the ChemiDoc MP Imaging System. Topics include, logging in, Smart Tray Technology, capturing your image, and exporting data.</p> </li> <li><a href="/en-us/category/image-lab-software-resources?ID=PJWA0VTU86LJ"><img src="/webroot/web/images/lsr/products/imaging_bioinformatics/category_feature/global/image-lab-video-th.gif" alt="Image Lab Software Tutorials" /></a> <h3><a href="/en-us/category/image-lab-software-resources?ID=PJWA0VTU86LJ">Image Lab Software Resources</a></h3> <p>Bio-Rad provides tutorials for basic acquisition and advanced analysis use of Image Lab Software 6.0.1. Topics include, densitometric analysis, molecular weight, normalization, purity assessment, and more!</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Application Guides</h3> <ul class="list-set list-three-col"> <li><a class="linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Western Blot Dr Troubleshooting Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-dr-troubleshooting.jpg" alt="Western Blot Dr. Troubleshooting Guide" /></a> <h3><a class="linkgeneration" onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Western Blot Dr Troubleshooting Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/en-us/applications-technologies/troubleshooting-western-blots-with-western-blot-doctor">Western Blot Doctor Troubleshooting Guide</a></h3> <p>Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look <span class="forcebreaks">their best.</span></p> </li> <li><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Protein Blotting Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-blotting-guide.jpg" alt="Protein Blotting Guide" /></a> <h3><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Protein Blotting Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_2895.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Protein Blotting Guide</a></h3> <p><span class="filesize">(PDF 7.9 MB)</span><br />Details on blotting technology, available products, and methods, plus tips, techniques, and troubleshooting.</p> </li> <li><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Electrophoresis Guide Img','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/western-blot-guides-electrophoresis-guide.jpg" alt="Electrophoresis Guide" /></a> <h3><a onmousedown="dcsMultiTrack('DCS.dcsqry','?tgp_pcp_link_group=Resources&amp;tgp_pcp_link=Electrophoresis Guide Txt','DCSext.tgp_pcp_link_group','','DCSext.tgp_pcp_link','','WT.dl','99999');" href="/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Electrophoresis Guide</a></h3> <p><span class="filesize">(PDF 6.4 MB)</span><br />A guide to polyacrylamide gel electrophoresis and protein detection, including theory, product selection, protocols, and more.</p> </li> </ul> <div class="dotteddivider dotteddividerdrk dotteddivider-less-top">&nbsp;</div> <h3 class="opt-subhead30">Documents Library</h3> <ul class="list-set list-two-col list-set-th"> <li><a href="/webroot/web/pdf/lsr/literature/Bulletin_6873.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/MA17-0012_S3_CV1_T2_01.png" /></a> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_6873.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">ChemiDoc MP System Brochure</a><br /><span class="filesize">(PDF 1.26 MB)</span></h3> <p>Explore the features and benefits of the new high-end imaging system for the best fluorescence and chemiluminescence detection.</p> </li> <li><a href="/webroot/web/pdf/lsr/literature/Bulletin_RP0051.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style"><img src="/webroot/web/images/lsr/products/electrophoresis/category_feature/global/MA17-0012_S3_CV1_T1_04.png" /> </a> <h3><a href="/webroot/web/pdf/lsr/literature/Bulletin_RP0051.pdf" target="_blank" rel="noopener noreferrer" class="no-target-style">Stain-Free Approach to Western Blotting</a><br /><span class="filesize">(PDF 864 KB)</span></h3> <p>Learn how to use stain-free technology for total protein normalization as an alternative to the standard blot normalization process.</p> </li> <li><a 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