Total Protein Normalization

Total protein normalization is the new standard required by journals for Western blot quantification.

Why normalize your Western blot

Normalizing a Western blot is crucial for accurate and reliable results in laboratory research. It accounts for variations in total protein levels due to experimental error, minimizing their impact on the final outcome. Normalizing increases accuracy, consistency, and the validity of results. You should perform proper normalization to enhance scientific rigor and contribute to knowledge advancement.

There are two ways to normalize your Western blot: single protein detection and total protein normalization. The single protein detection method uses housekeeping proteins (HKPs) and is often done by comparing the signal for the target protein to the housekeeping protein. A housekeeping protein is thought to be consistently expressed across different sample types. Its expression is believed to be unaffected by whatever intervention was carried out to produce the samples being compared.

Western blotting will tell you if your protein of interest is present in a sample. To determine if the amount of target protein is different between two or more samples, quantitative blotting is required (Table 1).

Quantitative Western blotting for TPN involves
Ensuring measurements of the protein of interest are within the linear range of the detection system used, and
Normalizing the measurements of the protein of interest to control for differences in the amount of sample loaded and/or any differences in transfer efficiency during the blotting protocol.

Table 1. Two factors of quantitative Western blotting

Common issues with single protein detection normalization and housekeeping proteins

HKPs are widely used as an internal reference for Western blot normalization. They are often highly expressed, lose proportionality at high protein loads, and only represent one portion of your whole sample. For this reason, using HKPs is an unreliable method of calculating normalization ratios.

Experimental conditions may change the expression of some commonly used housekeeping proteins. The expression level of the HPK must be similar to that of the protein of interest, otherwise one of the two proteins may not be within the linear range of detection. If the signal for the HPK is saturated under conditions needed to detect the target protein, it is not useful for normalization.

Why use total protein normalization over single protein detection

Fortunately, total protein normalization (TPN) can be used to measure the total protein abundance of each sample . This approach involves either

  • reversible staining (which occurs before immunodetection),
  • irreversible staining (which occurs after immunodetection), or
  • stain-free methods.

TPN provides a valuable opportunity for accurately evaluating protein abundance and can be tailored to meet specific research needs. 

Advantages of using Total Protein Normalization

TPN avoids the pitfalls associated with housekeeping protein normalization. In TPN, the signal for the protein of interest is normalized to the total amount of protein loaded in the lane. TPN provides a larger useful dynamic range, is not dependent on the expression of a housekeeping protein being unaffected by experimental manipulations, and can provide additional information about the quality of electrophoresis and Western blotting. It also takes into account:

  • Variations during electrophoresis
  • Variations during transfer
  • Sample loading variations
  • The intensity of all proteins in the lane

Which stains to use for total protein normalization

Total protein stains are used for TPN. You will find them easy to integrated into your existing Western blotting workflow. After transfer, the blot is stained with either a fluorescent or colorimetric stain, before blocking and antibody detection of the target protein.

Some stains are compatible with downstream detection. For example, AzureRed stain may be imaged with a 520nm laser, while one to three proteins may be detected using the other fluorescent channels (Figure 1).

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
Figure 1. 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.

TotalStain Q is compatible with downstream chemiluminescent or NIR detection. Combine it  with your downstream near-infrared or chemiluminescent imaging protocol for quantitative analysis, without any destaining step required. TotalStain Q accurately accounts for lane-to-lane variations inherent in Western blotting. With linearity up to 50 μg of lysate, it is ideal for the normalization of lowly expressed proteins that require high sample loads.

For ease of use, two kit varieties are available for PVDF or nitrocellulose membranes. Request a Sample >>

Watch this quick, 10-minute on-demand webinar using TotalStain Q for quantitative Western blotting results.

If you’re using other total protein stains, your Western blot may need to be imaged and destained before the blocking and antibody detection steps.

Our top tips for Total Protein Normalization

  1. Use enough stain/dye to completely submerse your blot
  2. Shake your blot while staining/destaining to evenly distribute stain/dye 
  3. Load lanes in duplicate or triplicate randomly across the gel
  4. Avoid edge effects. Dyes and stains can stain the edges of the blot more darkly than the interior of the blot
  5. Make sure to evenly load your gel
  6. Before loading, thoroughly mix samples and spin the contents to bottom of tube
  7. Establish range of linearity for each dye or stain
  8. Run a dilution series on a gel and stain to determine the range of protein that can be detected linearly

Imaging Systems for Total Protein Normalization

Azure Biosystems offers a full range of imaging systems, which include 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, treat your blots with TotalStain Q, between protein transfer and blocking, and process them as you would normally.

Azure Biosystems Sapphire FL for total protein normalization
The new Sapphire FL supports a broad range of excitation and emission wavelengths. We recommend the standard 532 optical module for detection of TotalStain Q.
Azure 300Q imager with green channel for total protein normalization
Add visible and/or NIR fluorescent detection capabilities as your needs evolve with the Q Module. The Azure 300Q imager is equipped with a green channel for total protein normalization.
Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers
Shown: Azure 500Q imager with green channel and 700nm and 800nm near-infrared lasers. You are able to add a Q Module for Total Protein Normalization of NIR Western blots using TotalStain Q. The upgrade Azure 500Q will also detect one more fluorescent target.

Compare imaging systems capable of imaging TPN here.

Frequently Asked Questions about Total Protein Normalization

For TPN with NIR Western blot detection, the Azure 500Q and the new Sapphire FL both provide detection of target analytes in the 700nm and 800nm channels, with a third channel used to measure TotalStain Q. Read more >>

The common practice for getting quantitative data from Western blots has been to normalize your band of interest to the signal from a housekeeping protein or to normalize to total protein. Read more about how to normalize your blot to total protein

Total protein normalization uses the entire protein content of each sample for normalization instead of relying on only a single housekeeping protein. Check out this example of total protein staining that shows detection of total protein in six identical samples of HeLa lysate before immunodetecting pSTAT3.

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Systems Equipped for TPN
Azure 500Q

Best solution for sensitive near-infrared and ECL Western blots

Sapphire FL Biomolecular Imager

Next generation laser-scanning system with unmatched flexibility and performance

Reagents for TPN

Additional Resources for Total Protein Normalization

Related Applications

Looking for a full list of applications?

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