Total Protein Normalization Bypasses the Problems with HKPs
The total protein normalization (TPN) method is better suited than HKP normalization for the detection of differences and changes in protein loads5 because it minimizes the influence of changes in the expression of individual proteins.6 TPN presents two major benefits over HKP normalization: (1) optimization is not required because total protein staining works across all tissue types and (2) overloading is less of a concern because of the wide linear dynamic range of some total protein stains.
Using TPN provides more accurate and reproducible results and is more cost- and time-effective than using HKPs. The experimental data from total protein normalization are more reproducible because the target protein level is directly correlated to the known total protein concentration in each lane. Researchers staining blots with total protein stains, such as Flamingo Pink, Sypro Ruby, Amido Black, or Ponceau S., have found that TPN exhibits excellent linearity at protein load quantities most commonly used in Western blotting.
Nevertheless, the TPN method too has its limitations. In particular, it does not possess the sensitivity to accurately quantify low amounts of total protein. Also it requires staining and de-staining procedures, adding a time and cost component and a process manipulation component that could variably impact sample signal and introduce experimental errors.4 In our hands the Ponceau S. stained-membrane must be visualized within 10 minutes because the signal intensity begins to drop after this time, usually resulting in poor-quality images and substandard quantification.4
For researchers that want to take total protein normalization one step further, a new approach has been recently introduced that enables normalization using total proteins without the staining step. This “stain-free” technology allows researchers to directly visualize and quantify proteins both in gels and on blots without the need to treat the blot, therefore providing significant time reduction, better accuracy, and cost-savings.7,9
The technology uses an in-gel fluorescent compound that irreversibly binds to tryptophan residues. As a result, the proteins can be fluorescently visualized in less than two minutes.10 The fluorescence is maintained throughout the Western blotting process so that proteins can be imaged at any point after separation.11
Compared to HKPs and TPN using stains, the stain-free technology better correlates to total protein concentrations, suggesting the higher sensitivity of the stain-free technology.4 Optimization of HKPs and staining steps are not needed, reducing the cost and time of the entire process from 16 hours (traditional HKPs) to six hours.10 It also provides a quality control check prior to the transfer and primary antibody steps, helping to save time and money.