Total Protein Staining for Protein-Expression Evaluation
Data generated from Western blotting can be misleading and full of inaccuracies if the proper loading and normalization controls are not accounted for. The change in a particular protein’s expression in response to a defined treatment, manipulation, or stimulus may be erroneously interpreted without a suitable point of reference. Relative quantitation of protein abundance is especially important for conveying the significance of observed variations, as proteins of interest frequently play key roles in physiological processes. To generate accurate and reproducible Western blot data, reliable protein loading standardization and normalization to a reference control are indispensable elements to your success.
The protein normalization component of Western blotting serves as an internal control, which requires an investigator-set baseline for relative comparison. In this way, protein expression changes are consistently quantitated across varying conditions, thereby ensuring the data are continuous and reproducible. How much of the observed change in protein expression (in response to x) can be attributed to an actual difference in the target protein’s abundance? To answer this question, housekeeping proteins are commonly employed as a point of reference. Such proteins have a characteristically consistent high-level of expression in all cells, and because of their maintenance, or housekeeping, duties, they are generally unaffected by experimental conditions.1 Housekeeping proteins such as actin, tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are examples of routinely used loading controls for lane-to-lane normalization. This is due to the fact that they are often steadily co-expressed with researchers’ protein(s) of interest.
Nevertheless, there are several drawbacks to their use, including potentially disproportionate expression between experimental conditions, membrane saturation due to their abundance in all samples, and reliance on a single data point for normalization.2-4 To account for these shortcomings, novel protein normalization methods that are more suitable and sensitive have emerged. Total-protein staining for data normalization is one such method that is quickly being adopted as an effective alternative.
Total-protein normalization is based on the sum of several or all band densities in one lane of the gel or membrane as a measurement of total-transferred protein. Unlike the housekeeping-protein normalization method, total-protein staining is a single-step, antibody-independent visualization process, obviating the potential for experimental manipulation and minimizing human error.
In addition to overcoming the limitations associated with the use of housekeeping proteins as loading controls, total-protein staining takes into account total protein transfer efficiency. During the Western blotting process, total protein can be observed at various stages using the appropriate gel or membrane stains, thereby serving as a continuous procedural control.2 The technique typically involves stain-based detection (e.g., Ponceau, Coomassie, silver, colloidal gold) after which the membrane or gel are immediately imaged for quantitative studies. Alternatively, stain-free technology that employs a proprietary polyacrylamide gel-embedded and ultraviolet light-dependent compound has recently become available. The advantage to this approach is that the covalently-bound and embedded fluorophore allows for the exposure and imaging of proteins at any point during the Western blotting procedure.
To obtain band density measurements, capturing an image of the membrane is the necessary first step, for which a digital imaging platform is preferred. Measurements are then recorded and further analyzed using compatible software. The sum of all bands per lane is then used for normalization against the band density values of the target protein(s), which are obtained in the steps that follow using specific primary and secondary detection antibodies.2 Summation, variation, and statistical calculations are frequently performed using multiple formulas and spreadsheets. Quantitative analysis is quickly becoming the norm for submissions to reputable, peer-reviewed scientific journals, but researchers are often conflicted on the path taken from the raw data to the analyzed, publish-ready results. To streamline these disaggrements, relative protein normalization calculations can be performed using a similar approach to that in qPCR data analysis, where fold change for each biological/technical replicate is determined as a fraction of the normalized loading control density.2 The basis of using biological/technical replicates for determining fold change allows for the calculation of averages and associated statistical analysis (p value), further strengthening the data.
Analytic consistency with protein abundance densitometry is key to trustworthy Western blot data. Reproducibility depends on the utilization of an appropriate and accurate baseline reference for relative normalization of changing target protein expression levels. Given the inherent weaknesses housekeeping proteins possess, total-protein staining offers a replacement detection and normalization method that permits more-precise quantitation of protein expression levels. The ease with which you achieve consistent Western blot data is of course also influenced by your proficiency in sample preparation, overall technique, and knowledge of available software and analysis platforms.
References
1. McDonough AA et al., “Considerations when quantitating protein abundance by immunoblot,” Am J Cell Physiol 308:C426-C433, 2015.
2. Posch A, Taylor CS, “The design of a quantitative western blot experiment,” Biomed Res Intl 2014:361590, 2014.
3. Greer S et al., “Housekeeping genes; expression levels may change with density of cultured cells,” J Immunol Methods 355:76-79, 2010.
4. Ferguson ER et al., “Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies,” Proteomics 5:566-571, 2005.