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Learn the methods for preparing 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.

A 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 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

Book Highlights

As with any quantitative study, consistency is the key to getting good results. Samples need to be handled the same way, and reagents, protocols, and workflows should be pre-validated and as identical as possible from run to run. But in addition to these general best practices, there are some steps you can take specific to chemiluminescent detection that will support generation of quantitative data.

1. Maximize your linear dynamic range by using a digital imager

Quantitation is only possible when the data you generate is linear, which is one of the drawbacks to using film for chemiluminescent detection. Fortunately, modern digital imagers far surpass the ~2-log dynamic range film offers. These features and more are available in the Azure Biosystems Imaging Systems and Sapphire™ Biomolecular Imager, including a dynamic range of up to ~6-logs.

2. Maximize your linear dynamic range with a long-lasting, quantitative ECL substrate

Not all ECL substrates are created equal. Azure’s Radiance ECL substrates—Radiance and Radiance Q—are designed with quantitation in mind. With a signal that persists for more than hours instead of minutes and a wide linear dynamic range, Radiance substrates are a great choice for quantitative chemiluminescent Western blots.

Overlay of four channels. Western blot stained with total protein stain, AzureRed, probed for three proteins of interest without a destaining step, scanned with Azure Sapphire Biomolecular Imager
AzureRed is imaged simultaneously with three proteins of interest. The gel was loaded with dilutions of HeLa cell lysate. After transfer, the blot was stained with AzureRed and then probed for tubulin, ß-actin, and GAPDH without a destaining step. The blot was scanned with each of the four lasers of the Sapphire Biomolecular Imager. In this overlay of the four channels, total protein (AzureRed stain) is shown in gray; tubulin in red, ß-actin in blue, and GAPDH in green.
3. Normalize chemiluminescent westerns to total protein​

With journals and western blot experts recommending that blots be normalized to total protein rather than a housekeeping protein 1-4, normalizing chemiluminescent Western blots no longer requires stripping and re-probing the blot or running multiple blots if you use AzureRed Total Protein Stain.

AzureRed Total Protein Stain is an efficient and robust way to normalize data for quantitative chemiluminescent Westerns. AzureRed is compatible with both fluorescent and chemiluminescent detection.

Make sure you're 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 using an Azure imaging system.

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.
Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager

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