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.