Problem: Reliably quantifying proteins in western blot analyses can be challenging even for seasoned researchers. This is partly due to challenges associated with the use of housekeeping proteins (HKP) as controls in normalization.
For quantitative western blot, data normalization is needed to account for inconsistencies in sample preparation, sample loading, and uneven protein transfer to provide more accurate and reproducible results. Different normalization methods, in theory, should provide the same result: the amount of the target protein(s) in a sample, relative to other samples in the same gel or blot.
HKPs can be useful as an internal loading control or a proxy for the entire protein population. When using these, researchers have to do three things: show that the HKP is expressed in the cell or tissue of interest and include a control lysate which does not express the HKP; ensure HKP expression levels are consistent across experimental conditions; and determine the linear quantitative range of the HKP. These steps are necessary for producing publishable work with HKPs but are difficult and time-consuming.
Solution: A more efficient and accurate alternative to HKPs exist for western blot normalization. Researchers can normalize western blots using the total protein in the sample, known as total protein normalization (TPN). In TPN, the abundance of the target protein is normalized to the total amount of protein in each lane. Because TPN is not dependent on a single loading control, validation of controls and stripping/reprobing of blots for detection of HKPs is not necessary.
In TPN, scientists can measure the amount of total protein in a gel or blot by staining, but a preferred stain-free process is also an option. Stains may not uniformly cover the gel or blot, which affects accuracy and reproducibility. The intensity of some stains such as Ponceau S are also time-dependent. The stain-free method, developed by Bio- Rad, does not have such drawbacks. It employs an in-gel chemistry that allows proteins throughout the gel or blot to be detected during imaging. Eliminating stain-destain steps also saves time.
Stain-free TPN also has the advantage of low background levels, which can improve sensitivity, down to 0.1 μg of total protein per lane. Total protein stains require additional staining time to visualize strong protein bands, which results in high background signal from unwashed stain. Stain-free technology enables visualization of strong protein bands with low background signals from gels.
To learn more about TPN, please visit Bio-Rad Laboratories’ two-part webinar series on western blot normalization at bio-rad.com/TPNwebinar. This webinar series discusses similarities and differences between HKPs and TPN methods, and provides a step-by-step guide for accurate quantification of proteins using stain-free gels.