How to Normalize Western Blots to Total Protein

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Table of Contents

Why normalizing to a housekeeping protein is not always the best method

A staple of many life science labs, the Western blot has evolved from the humble off-shoot of DNA and RNA blotting methods into the go-to technique for identifying specific proteins in a complex mix, verifying protein identity, and determining relative protein amounts. It’s easy, inexpensive, and the necessary instruments and reagents are widely accessible (we’re looking at you, mass spectrometry).

One recent improvement to the technique impacts how we perform quantitative Western blotting—specifically, how we normalize bands on the blot. The new recommendations to normalize to total protein instead of to a housekeeping protein should lead to Western blot data that is more accurate and reproducible.

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.

Why Normalize to Total Protein

The common practice for getting quantitative/semi-quantitative data from Western blots has been to normalize your band of interest to the signal from a housekeeping protein, the assumption being that being essential, the abundance of specific housekeeping proteins would be invariant across tissues and conditions. 

“Over the past two decades, it became clear that this assumption [about housekeeping proteins is essential] is wrong.”

Since as early as 2014², scientists have been concerned about the use of housekeeping proteins for normalizing Western blots. At that time, a number of studies showed that many of the proteins commonly used for normalization, such as GAPDH, tubulin, and actin, are expressed at levels that can vary between tissue types and experimental conditions. The implications for past Western blot studies is staggering. It’s clear that moving forward researchers either need to use another normalization method to get accurate, reliable, and reproducible quantitative Western blot data or else exhaustively verify that the abundance of the protein being used for normalization remains invariant across the tested conditions and is present at similar levels as the protein-of-interest.

Another objection to using housekeeping proteins is the observation that many of them are present in much higher abundance than the protein-of-interest and, thus, are likely to be outside of the linear dynamic range of the blot1.

Total Protein Normalization is the New Normal

The emerging consensus on the best way to normalize Western blots is to normalize to total protein. Many journals have embraced total protein normalization (TPN) and some, such as the Journal of Biological Chemistry, even require authors to use TPN when publishing quantitative data from Western blots or else to validate the use of their housekeeping protein3, 4.

Overview of Total Protein Stains (TPS) and the TPN workflow

There are a range of total protein stains to choose from (see Mortiz1 for a nice overview of different TPS options). The TPS you choose will affect the complexity of the TPN workflow.

Important factors to consider when choosing a TPS:

  • Dynamic range
  • Detection limits
  • Visualization method
  • Staining time
  • Visualization time
  • Consistency across tissues and experimental conditions
  • Compatibility with antibody-based detection

Not all stains are alike and some stains are easier to use and more accurate than others. For example, there are stains which are used on the protein gel, which sounds straight-forward but can impact the efficiency of transfer to the membrane and, thus, quantitation and reproducibility. Other stains are used after transfer so will (obviously) have no impact on transfer efficiency for better accuracy and reproducibility. If your visualization instrument is limited to two channels for detection, you will need to strip and re-probe the blot to evaluate multiple proteins, which does reduce accuracy and reproducibility.

AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins.
AzureRed showed superior correlation and a much broader dynamic range than the common housekeeping proteins, such as GAPDH.

Total protein normalization workflow using AzureRed Total Protein Stain

We are dedicated to developing products that have a large positive impact on your work while having a minimal impact on your current workflow. AzureRed Total Protein Stain is just one example. With minimal disruption to the typical Western blot workflow—there’s an additional wash/incubation step before membrane blocking—you can easily stain for (and normalize to) total protein. Using AzureRed is simple, consistent, accurate, and flexible. More benefits to using it are listed in Table 1.

Table 1. Perks to incorporating AzureRed into your current Western blot workflow

AzureRed is...
Simple to useReversible and compatible with downstream antibody-based detection
ConsistentDelivers a signal that’s reproducible and unaffected by tissue-type and experimental conditions. It is designed to preserve a consistent pH level, aiding the elution of proteins from the gel, as well as their binding to the membrane
AccurateLinear over a wide dynamic range (> 3-log) for robust quantitation
FlexibleCan be used with fluorescently-labeled antibodies, as well as the Azure Imagers or other chemiluminescent detection systems

The AzureRed workflow (Figure 2) is simple and adds minimal time to the Western blot protocol. Excitation is via the 520 nm/Cy3 channel on the Sapphire Imager.

Since the release of this blog post, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more.

(Left Panel) Image of a fluorescent multiplex Western blot with three proteins of interest using AzureRed. Imaged on the Azure Sapphire Biomolecular Imager. (Right Panel) AzureRed total protein stain has a wider linear dynamic range compared to common housekeeping proteins.
Figure 2. TPN workflow using AzureRed Total Protein Stain. (Left Panel) AzureRed is imaged simultaneously with three proteins of interest. Imaged on the Sapphire Biomolecular Imager. (Right Panel) AzureRed has a wider linear dynamic range compared to common housekeeping proteins.

Using the Cy3 channel keeps the NIR channels available for detection of multiple proteins, without needing to strip and re-probe the Western blot. You can also remove AzureRed after staining as it is a removeable stain.

AzureRed Total Protein Staining Short Protocol

1. Washing
  • Following transfer, wash blot for 5 min in water.
  • Proceed to PVDF (2) or Nitrocellulose (3) protocol.
2. PVDF Protocol
  • 2a. Staining
    • Place blot protein side down in Stain Solution
    • Stain blot with gentle rocking for 15–30 min
  • 2b. Acidification
    • Place blot in Fix Solution and incubate with gentle rocking for 5 min.
  • 2c. Wash
    • Rinse blot 3 times with 100% ethanol for 2–3 min each, until green background on blot has been completely removed
  • 2d. Drying
    • Hang blot from a peg or dry on wire mesh to allow blot to dry evenly. Allow blot to dry completely before imaging

Note: The above is an abbreviated protocol. You can find the full protocol including how to use AzureRed for staining gels and how to remove it afterwards by downloading our free Western Blotting eBook to learn the best tips and tricks for Western blotting.

FREE WESTERN BLOT eBOOK

New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!

Additional blog posts on total protein:

Shop AzureRed and Reagents for Normalizing to Total Protein

SOURCES

  1. Mortiz CP. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics. 2017 Oct; 17(20). PMID: 28941183 (includes
    link to free full text).
  2. Ghosh R, Gilda JE, Gomes AV. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics. 2014 Oct;11(5):549-60. PMCID: PMC4791038.
  3. Collecting and presenting data. The Journal of Biological Chemistry website. http://jbcresources.asbmb.org/collecting-and-presenting-data#blot. Accessed February 4, 2019.
  4. Fosang AJ and Colbran RJ. Transparency Is the Key to Quality. J Biol Chem. Dec. 11 2015. 290(50). 29692–29694. PMCID: PMC4705984.

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