Total Protein Normalization Stain

Western Blotting

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    How to Normalize Western Blots to Total Protein

    And why normalizing to a housekeeping protein can lead you astray

    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.

    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. As Christian Moritz succinctly states in a 2017 Proteomics review1:

    “Over the past two decades, it became clear that this assumption 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, and 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 Now 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.

    An overview of the Total Protein Stains (TPS) and the TPN workflow

    There are a range of total protein stains (TPS) 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, and the important factors to consider when choosing a TPS include:

    • 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. But 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.

    The TPN workflow using AzureRed Total Protein Stain

    At Azure™ Biosystems, we are dedicated to developing products that have a large positive impact on a scientist’s work while having a minimal impact on a scientist’s workflow. Our AzureRed Total Protein Stain is one example. With minimal disruption to a typical western blot workflow—there’s an additional wash/incubation step before membrane blocking—you can easily stain for (and normalize to) total protein. The process is:

    • Simple—AzureRed is a reversible stain that is compatible with downstream antibody-based detection
    • Consistent—AzureRed delivers a signal that’s reproducible and unaffected by tissue-type and experimental conditions
    • Accurate—AzureRed is linear over a wide dynamic range (> 3-log) for robust quantitation
    • Flexible—AzureRed can be used with fluorescently-labeled antibodies as well as chemiluminescent detection systems
    The 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.

    The workflow is simple and adds minimal time to the western blot protocol, and excitation is via the 520 nm/Cy3 channel, keeping NIR channels available for detection of multiple proteins without needing to strip and re-probe the blot.

    AzureRed Total Protein Staining Protocol

    Note that this is an abbreviated protocol. You can find the full protocol including how to use AzureRed for staining gels and how to remove AzureRed stain (AzureRed is a reversible stain) by ordering AzureRed Total Protein Stain or by downloading our Western Blotting Guidebook.

    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


    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 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.


    Western blot normalization: Housekeeping protein vs total protein

    Using Housekeeping Proteins for Normalization

    The most significant drawback of using housekeeping proteins is that their levels may not be consistent across samples and conditions.1,2 It is possible to use a housekeeping protein for normalization, but you must first spend the time and effort to validate your choice, and may 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.

    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

    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.

    Finally, 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 blot. This becomes increasingly difficult when an experiment examines multiple proteins of interest on the same blot.


    Using Total Protein Staining 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 and this value is used as the denominator when normalizing.1-4 Many total protein stains are available that can be used to stain gels and membranes.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 be much faster than using a housekeeping protein, especially for chemiluminescent blots because the staining step takes less time that stripping and reprobing the blot. Ideally, total protein staining is conducted on the membrane, either before or after immunodetection.2 With 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.  With this simplest of workflows, images for the protein(s) of interest and total protein are automatically aligned, avoiding the need resize and align images captured at different times.

    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.

    Looking for more information about Western blot normalization?
    Read more about using AzureRed Fluorescent Total Protein Stain for total protein normalization of fluorescent Western blots by clicking below.


    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.


    Document TypeDescription
    Application NoteAccurate Western Blot Normalization with AzureRed Fluorescent Protein StainDOWNLOAD