Guide to Total Protein Normalization in Western Blotting

For years, scientists have used housekeeping proteins (HKPs) as loading controls for western blot normalization. However, this approach is being superseded by total protein normalization, which offers greater experimental consistency and improved laboratory throughput. This article explores some of the different methods that have been developed for total protein normalization and explains how these can be integrated into your workflow.

Limitations of housekeeping proteins for western blot normalization

Housekeeping proteins such as actin, tubulin, and GAPDH are essential for basic cell functions like intracellular transport, cell division, and energy production. As such, they are expressed constitutively at high levels—a characteristic that has seen HKPs widely used as loading controls for western blot normalization. But HKPs have increasingly fallen out of favor for western blot applications due to several inherent shortcomings.

“Studies conducted at Thermo Fisher Scientific have demonstrated that HKPs are prone to signal saturation, can produce inaccurate normalization results, and may exhibit sample-to-sample variability approaching 50%,” explains Thomas Diller, Ph.D., Thermo Fisher Scientific Staff Scientist. In addition, Pillai-Kastoori et al. have reported multiple limitations associated with HKP-based normalization. These include variable expression of HKPs across different cell lines and tissue types, altered HKP expression under varying experimental conditions and cell culture densities, and masking of proteins of interest, which can complicate accurate visualization and quantification.^1,2

For these reasons, the use of total protein normalization for western blotting is becoming more common—and is increasingly required by academic publishers and grant agencies. Indeed, as standards for reproducibility and quantitative rigor have increased, many journals across the life sciences now expect authors to justify normalization strategies and avoid unvalidated reliance on HKPs. Some journals, like the Journal of Biological Chemistry, even caution against the use of HKPs for normalization in their Instructions to Authors.

Advantages of total protein normalization

“A major advantage of total protein normalization is that it improves data accuracy and reproducibility by directly measuring the entire protein signal in each lane rather than relying on a single housekeeping protein,” reports Deanna Woo, Product Manager, Protein Quantitation Business at Bio-Rad. “Additionally, total protein normalization solutions such as Bio-Rad’s proprietary Stain-Free™ technology eliminate the need to perform staining and destaining steps, shortening the workflow by 50% of the time compared to traditional western blotting.” Other advantages of total protein normalization are that it has the potential to cut costs by reducing reagent use and repeat experiments, and can increase the likelihood of manuscripts being accepted for publication.

Total protein normalization technologies

Technologies for total protein normalization vary in terms of how they work. The following are three leading examples:

• Stain-Free™ technology (Bio-Rad)

Bio-Rad’s Stain-Free technology is based on trihalo compounds incorporated directly into TGX Stain-Free gels that, upon brief UV activation, react with tryptophan residues in proteins to generate a stable fluorescent signal.³ “Researchers also have the option to use TGX Stain-Free™ FastCast™ Acrylamide Kits for hand casting,” says Kenneth Oh, Ph.D., Marketing Manager, Protein Quantitation Business at Bio-Rad. “Our approach eliminates the need for post-electrophoresis staining and allows proteins to be visualized in-gel, on the blot post-transfer, and throughout the workflow using a Bio-Rad Stain-Free enabled imaging system, such as the ChemiDoc family of imaging systems or the GelDoc Go. With a broad dynamic range that is less prone to saturation than using HKPs, and an intuitive method, Stain-Free technology enables users to generate more accurate and reliable quantitative data, faster.”

• Amersham™ QuickStain Protein Labeling Kit (Cytiva)

Amersham QuickStain detects the total protein content of a sample through Cy5 NHS ester labeling of reactive amino groups.⁴ A second dye is used to detect the target protein. “Researchers simply dilute their samples in the optimized labeling buffer and add a fixed amount of Cy5, then incubate for 5 minutes at 95°C for qualitative analysis or 30 minutes at room temperature for quantitative applications,” says Meena Ali, Ph.D., Global Product Manager for Western Blotting Consumables at Cytiva. “Key features of QuickStain include its broad, linear dynamic range, which allows simultaneous quantification of major bands and low‑level impurities, and its high sensitivity, which can detect impurities down to 0.1% of the main band using a fluorescence scanner or imager. QuickStain is compatible with self-poured gels and a variety of commercial gels and allows for total protein measurement on any membrane without relying on UV imaging or low fluorescence PVDF.”

• No-Stain™ Protein Labeling Reagent (Thermo Fisher Scientific)

Thermo Fisher Scientific’s No-Stain Protein Labeling Reagent covalently labels lysine residues, which are abundant across the proteome. It can be imaged with UV, green or blue LED excitation, making it compatible with most western blot imagers, including the Invitrogen™ iBright™ FL1500 Imaging System*. Notably, when acquiring an image of a No-Stain-labeled membrane with green or blue LED excitation, there is less background autofluorescence from the membrane, which improves signal strength and quality. “Advantages of No-Stain include a fast and easy labeling protocol, broad compatibility with multiple gel chemistries, and the ability to be imaged on most western blot imagers,” says Thompson. “No-Stain also allows users to label either the gel or membrane directly, depending on their application, and benefits from tunable sensitivity, whereby the labeling time or labeling reagent concentration can be increased to produce a stronger signal.”

Conclusion

Total protein normalization is widely acknowledged to be a more robust and accurate method for western blot normalization than using housekeeping proteins, whose expression can easily saturate and can vary with cell type, treatment, or experimental conditions. Many different technologies for total protein normalization are now available, enabling researchers to streamline their western blot workflow without adding complexity.

References

1. Diller T, Thompson J and Steer B. Biological validation of a novel process and product for quantitating western blots. Journal of Biotechnology, 326, 52–60 (2021).

2. Pillai-Kastoori L, Schutz-Geschwender AR and Harford JA. A systematic approach to quantitative Western blot analysis. Analytical Biochemistry, 593, 113608 (2020).

3. Short R and Posch A. Stain-Free Approach for Western Blotting: Alternative to the Standard Blot Normalization Process. Genetic Engineering & Biotechnology News, Vol. 31, No. 20 (2011).

4. Hagner-McWhirter Å, Laurin Y, Larsson A, Bjerneld EJ, Rönn O. Cy5 total protein normalization in Western blot analysis. Anal Biochem. 486:54-61 (2015).

* For Research Use Only.