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Quantitative Western blots

quantitative western blot basics

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A quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide includes a troubleshooting section and tear-out quantitative Western blotting checklist.

Are you following the right steps to preparing a quantitative Western blot?

Western Blot imaged with Azure Imager

Getting quantitative data from your Western blot takes more than just a fluorescent secondary and digital imager. You need to consider your sample prep, your normalization method, and the linearity of your signal. To help keep your quantitative Western blot workflows on track, we’ve created a checklist of steps that can be taken to ensure your Western blot quantitation is as accurate as it can be.

Featured Publication

Check out this publication from Boys Town National Research Hospital that imaged chemiluminescent Western blots with an Radiance HRP Substrate and an Azure imager.

Use this Checklist for Quantitative Western Blotting

Consistency is key!

Have you treated your samples consistently, ensuring cells and tissues are thoroughly lysed, immediately placed on ice, and/or been treated with appropriate reagents such as protease inhibitors?

Have you treated your samples consistently, ensuring cells and tissues are thoroughly lysed, immediately placed on ice, and/or been treated with appropriate reagents such as protease inhibitors?

Are you using a robust and consistent western blotting protocol (remember that you can reduce variability by using the same reagents for each blot or implementing lot-to-lot controls, and keeping your transfer conditions and incubation and wash times the same)?

Are you normalizing to total protein?

Have you validated the specificity of your antibodies?

Have you verified that your system is linear?

  • Have you treated your samples consistently, ensuring cells and tissues are thoroughly lysed, immediately placed on ice, and/or been treated with appropriate reagents such as protease inhibitors?

  • Have you loaded similar amounts of total protein in each lane (within a factor of 2- to 5-fold)?

  • Are you using a robust and consistent western blotting protocol (remember that you can reduce variability by using the same reagents for each blot or implementing lot-to-lot controls, and keeping your transfer conditions and incubation and wash times the same)?

  • Are you normalizing to total protein?

  • Have you validated the specificity of your antibodies?

  • Have you verified that your system is linear?

The linearity of your signal can be affected by multiple factors. Here are steps you can take to ensure linearity and improve the accuracy of your quantitation:

  • Are the highest and lowest amounts of your protein-of-interest in the linear range (note that you need to verify linearity with each protein-antibody pair)?

  • Are you saturating your membrane?

  • Have you optimized your primary and secondary antibody dilutions? Titrating your antibody against your protein-of-interest can reveal an antibody dilution that delivers the widest dynamic range.

  • Have you optimized your image acquisition time to ensure you are in the linear range of your imager?

Use reliable reagents that support accurate quantitation

Workflow comparing traditional western blot work flow to the workflow for quantitative western blots

Complete the form below to get the Quantitative Western Blotting Basics Guide.

    Powerful and Flexible Western Blot Imagers

    With products that offer sensitive detection, wide dynamic range, easy-to-use features, and lot-to-lot consistency Azure Biosystems expertly streamlines and supports your quantitative fluorescent Western blot workflows.

    • Choose an Azure Imager when you need an instrument that delivers it all—performance, bench-friendly footprint, and affordability. Available in a range of upgradable configurations that support imaging with NIR
      fluorescence using lasers, RGB fluorescence (LEDs), chemiluminescence, and UV and white light.
    • Choose the Sapphire Biomolecular Imager when you need a scanning imager or need the highest performance levels. Available in a range of upgradable configurations that support scanning/imaging with NIR fluorescence (lasers), RGB fluorescence (lasers), and phosphor imaging capabilities.
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