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.
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 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. 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). AzureRed allows you to stain 1D and 2D gels in less than 3 hours, with high sensitivity, low background, and no speckling.
Benefits and challenges of using a housekeeping protein vs. total protein for Western blotting
|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|
– Familiar, 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 resolved from protein of interest on gel
– Larger linear dynamic range
– Low variability
– Constant across sample types
– No change with experimental conditions
– Must ensure total protein stain used is compatible with antibody binding and detection method
Challenges to using Housekeeping Proteins for Normalization
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.
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
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.
Use the links below to give AzureRed a try for your next Western blot. While you’re here, check out our brochure for the complete overview of the total protein stain options available from Azure. For how to perform Accurate Western Blot Normalization with AzureRed Fluorescent Protein Stain, this application note is also available. Cheers!
Shop Total Protein Stains
AzureRed Fluorescent Total Protein Stain
Azure Ponceau Stain
TotalStain Q (NC)
TotalStain Q (PVDF)
Frequently Asked Questions
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.
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
- Moritz CP. Tubulin or not tubulin: heading toward total protein staining as loading control in Western blots. Proteomics. 2017;17:1600189.
- 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.
- McDonough AA et al. Considerations when quantitating protein abundance by immunoblot. Am J Cell Physiol. 2015;308(6):C426-C433.
- Fosang AJ, Colbran RJ. Transparency is the key to quality. J Biol Chem. 2015;209(50):29692-29694.