How to Normalize Western Blots to Total Protein

Western Blotting

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.


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


  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. 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.

Quantitative Westerns: What is the Best Way to Normalize your Western blot?

Fluorescence imaging Multiplex Quantification Western Blotting

Far from being an “is-it-there-or-not” technique, modern digital detection instruments can make Western blotting reproducible and quantitative. By working within the linear dynamic range of your detection method and normalizing the data to control for variations in protein load and membrane transfer, you can get truly quantitative results.

But what is the best way to normalize protein levels for a Western blot? In the past, the gold standard normalization method was to use a housekeeping protein based on the assumption that the levels of these proteins are fairly consistent across experimental conditions and cell lines. However more recent studies have shown that this assumption is not always true1,2 leading to inaccurate measurements of relative protein abundance. Instead, quantitative Western blotting experts1,2 and the journals they publish in4 are recommending a new gold standard for normalization—normalizing to total protein detected in each lane, preferably by staining on the membrane.

Table of contents

Using Total Protein Stains for Normalization

With total protein normalization, instead of trying to find a protein that can represent the total amount of sample that transferred to the membrane, total protein is measured on the membrane directly. This value is then used as the denominator when normalizing.1-4 Many total protein stains used to stain gels and membranes are commercially available.1 Total protein stains provide a larger dynamic range and demonstrate lower variability and cleaner data than housekeeping proteins.1,2

Total protein normalization can also be much faster than using a housekeeping protein, especially for chemiluminescent Western blots. This is because the time it takes to stain the blot takes less time compared to stripping and reprobing. Ideally, total protein staining is conducted on the membrane, either before or after immunodetection.2 Using some stains, such as AzureRed Fluorescent Total Protein Stain, it is possible to stain the blot before immunodetection and then to image total protein simultaneously with the protein(s) of interest. For Western blots and gels, AzureRed is able to detect less than 1 ng of protein per band or spot and is non-toxic and biodegradable, for safe and simple disposal.

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.

AzureRed is a quantitative, fluorescent protein stain for total protein normalization in blots and total protein detection in gels. It is fully compatible with downstream Western blotting or mass spectrometry.

AzureRed Fluorescent Total Protein Stain
Choose AzureRed Fluorescent Protein Stain for sensitive detection of total protein on 1D or 2D gels. AzureRed is as sensitive as silver stain and is compatible with downstream Western blotting, mass spectrometry, and Edman sequencing. It provides very low background and higher signal to noise than other fluorescent stains and can be imaged with UV or blue light excitation.

AzureRed allows you to stain 1D and 2D gels in less than 3 hours, with high sensitivity, low background, and no speckling. Stained gels and blots can be imaged on both the new Sapphire FL (or other laser-based systems) and the Azure Imaging Systems (or other CCD-based fluorescent imaging systems).

Advantages and disadvantages with using Housekeeping Proteins for Normalization

Housekeeping protein• Familiar and commonly used• Narrow, linear dynamic range • Abundance can vary with experimental conditions • Abundance may not be consistent between sample types • High variability • Must ensure housekeeping protein physically resolves from protein of interest on the gel
Total protein• Larger linear dynamic range • Low variability • Constant across sample types • No change with experimental conditions• Must ensure the total protein stain that's used is compatible with antibody binding and detection method

Table 1. Benefits and challenges of using a housekeeping protein vs. total protein for Western blotting

Inconsistent levels

The most significant drawback of using housekeeping proteins is their levels may not be consistent across samples and conditions.1,2 While it is possible to use a housekeeping protein for normalization, but you must first spend the time and effort to validate your choice. You may also need to examine multiple potential standards before you find one that is truly expressed at the same level across all of your samples and does not change across your experimental conditions.

High abundance

A second significant challenge associated with housekeeping proteins is their high abundance.1,3 If the housekeeping protein is present at a very high level in your sample, this limits the amount of sample you can load on the gel because you will need to keep the housekeeping protein within the linear range of detection and not saturate the signal for the housekeeping protein. This is particularly problematic if the protein of interest is not similarly highly expressed, because the two proteins will not be within the same linear range of detection.2,3

Generating primary and secondary antibodies from non-overlapping species is difficult

A third challenge to consider if you’re doing multiplex Western blots, such as comparing phosphorylated and non-phosphorylated forms of the same protein, is the complexity of generating primary and secondary antibodies from non-overlapping species.

Keep in mind it is always possible that detecting the housekeeping protein could interfere with detection of the protein of interest.1 Ideally, the housekeeping protein should be a different size than the protein of interest, so the two proteins are spatially resolved on the Western blot. This becomes increasingly difficult when an experiment examines multiple proteins of interest on the same Western blot.


>> ADDITIONAL READING: Multiplex fluorescent Western blotting

Analysis after using a total protein stain

Comparison of a traditional western blot workflow to a western blot workflow using AzureRed Total Protein Stain
Comparison of a traditional Western blot workflow to a Western blot workflow using AzureRed Total Protein Stain

The analysis workflow after image capture is essentially unchanged compared to using a housekeeping protein; the signal density for the entire lane or a large portion of the lane is used for normalization instead of the density for a single band.

Staining the membrane with a total protein stain provides an added quality control benefit, allowing verification that membrane transfer was complete and free of artifacts. With this very simple workflow, images for the protein(s) of interest and total protein are automatically aligned, avoiding the need resize and align images captured at different times.

While you’re here, check out this brochure for a complete overview of available total protein stain options. For more on how to perform accurate Western blot normalization using AzureRed Fluorescent Protein Stain, check out this application note. Cheers for now.

Additional blog posts regarding total protein:

Frequently Asked Questions

Total protein normalization (TPN) is used to quantify the abundance of the protein of interest, without having to rely on housekeeping genes. It is usually done by incubating the membrane with a total protein stain. Read more

TPN uses the entire protein content of each sample for normalization instead of relying on only a single housekeeping protein. You can see an example of total protein staining here.

AzureRed is a perfect choice for staining applications, including post-transfer staining to confirm uniform protein transfer from gel to membrane, and
staining quantitative Western blots as part of a TPN protocol. Read more

Azure offers a range of imaging systems includes several models that allow target protein detection to be multiplexed with TPN – with no need for dedicated precast gels or laborious stripping and re-probing. Instead, you simply treat your blots with TotalStain Q between protein transfer and blocking, and process them as you would normally. Read more

Shop Total Protein Stains


  1. Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. Proteomics. 2017;17:1600189.
  2. Thacker JS et al. Total protein or high-abundance protein: which offers the best loading control for Western blotting? Anal Biochem. 2016;496:76-78.
  3. McDonough AA et al. Considerations when quantitating protein abundance by immunoblot. Am J Cell Physiol. 2015;308(6):C426-C433.
  4. Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;209(50):29692-29694.