Normalization is a critical step in attaining accurate data from quantitative western blots. By correcting for variations in sample preparation, protein loading, and transfer efficiency across a membrane, normalization ensures that changes in band intensities can be correlated to biological differences between samples. Historically, antibody-based detection of housekeeping proteins (HKPs) was the preferred method for normalizing western blot data. However, total protein normalization (TPN) is now widely acknowledged to be a superior approach that delivers more reproducible results. This article explains the basic principles of total protein normalization and suggests ways of incorporating TPN into your quantitative western blot workflow.
Common housekeeping proteins such as β-actin, tubulin, and GAPDH were once thought to exhibit constant expression levels, resulting in their widespread use as controls for normalizing western blot data. However, it is now known that HKP expression varies across different cell lines and tissue types (Figure 1) and can be influenced by experimental conditions, which introduces significant potential for error. A further drawback of using HKPs for western blot normalization is that they are usually highly expressed, whereas target proteins are often expressed in lower abundance. Thus, to detect the target of interest, it is frequently necessary to load large amounts of cell lysate, resulting in oversaturated HKP reference bands that are out of their linear range.
Figure 1. Differences in three candidate housekeeping proteins between tumor (T) and non-cancerous (N) tissues in a validation sample set. Immunodetection measurements of HKP levels show poor linearity and do not accurately indicate cell lysate loading levels.1
Instead of using just a single housekeeping protein to normalize western blot data, TPN uses the combined signal intensity from all proteins in the same sample. Established methods for TPN involve incubating the membrane in a total protein stain, such as Ponceau S or Amido Black, and capturing an image of the protein bands before proceeding with antibody-based immunodetection. Because total protein stains are less sensitive than antibody-based detection of HKPs, they exhibit good linearity in the common loading range of 10–50 μg of cell lysate and are far less likely to result in an oversaturated signal. However, the use of total protein stains typically requires tedious staining and destaining procedures and can potentially interfere with subsequent blot development and visualization.
The inability to replicate experimental data is an ongoing challenge for the scientific community. To help address this problem, several major scientific journal publishers have recently revised their editorial guidelines for data publication—including the introduction of more stringent requirements for quantitative western blotting. For example, the Journal of Biological Chemistry states that “housekeeping proteins should not be used for normalization without evidence that the experimental manipulations do not affect expression” and recommends that signal intensity should be normalized to total protein loading whenever possible.2 As such, researchers are increasingly incorporating TPN into their western blotting experiments.
To make total protein normalization both quicker and easier, various alternatives to conventional total protein stains have been developed. These include Bio-Rad’s Stain-Free imaging technology, which uses a proprietary trihalo compound in TGX Stain-Free gels. Upon brief UV activation, the compound covalently modifies tryptophan residues, causing them to fluoresce and enabling visualization of total protein. With Stain-Free technology, researchers can capture images of the gel and membrane before and after transfer (Figure 2) without having to perform laborious staining and destaining steps, potentially saving time by identifying any problems early in the western blot workflow. Image Lab software streamlines western blot analysis by facilitating quantification, TPN, and data analysis.
Figure 2. Verification of protein transfer using a stain-free enabled imaging system. Serial 1:2 dilutions of hemoglobin (starting quantity, 80 ng), with 1.8 μg of BSA/lane as a carrier (top band), were electrophoretically separated on a 4–20% 26-well Criterion Stain-Free gel. Images were taken using the Criterion Stain-Free imager.
Compared to the customary practice of using housekeeping proteins for normalizing quantitative western blot data, total protein normalization provides more accurate and reproducible results. Although TPN can be accomplished using total protein stains like Ponceau S or Amido Black, alternative approaches such as Bio-Rad’s Stain-Free imaging technology make TPN quicker and easier, helping researchers to more readily meet the new editorial guidelines for data publication. To learn more about Stain-Free imaging technology, visit bio-rad.com/stainfree
1. Hu X, Du S, Yu J, et al. Common housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better "housekeeper". Oncotarget. 2016;7(41):66679-66688.
2. https://jbcresources.asbmb.org/collecting-and-presenting-data