Aldrin V. Gomes, Ph.D.
Ning Liu Ph.D.
A closer examination of housekeeping and other normalization techniques.
In recent years, the scientific community has questioned the reliability of Western blotting as a quantitative method for measuring relative protein levels.1 This scrutiny largely stems from the lack of standardization and reproducibility in the Western blotting process.
Inherent process inconsistencies, such as sample preparation, gel loading, and protein transfer must be accounted for. Variations in protein transfer efficiency—an often overlooked variable—can result in a two- to four-fold increase or decrease in signal between gel lanes.1 To correct for these nonsample-related effects on signal intensity, researchers apply normalization techniques.
The traditional and most frequently used method for normalizing Western blotting results is the use of loading controls—namely, housekeeping proteins (HKPs). The most commonly used HKPs include tubulin, GAPDH, and actins. These proteins are known to be present in high quantities within biological samples. The protein quantities are assumed to be fairly constant across most cell types and remain relatively insensitive to environmental conditions and treatments.
Although the HKPs listed above are more conserved between different cell types, they are not always expressed at constant levels. Several publications suggest that HKPs are cell and sample-treatment dependent2,3. This raises two major concerns when using housekeeping proteins for normalization, namely, the overloading of HKPs and the fact that HKP levels depend on cell type and condition. Not accounting for these factors may jeopardize the validity of your Western blot results.
The Limits of Housekeeping Protein Normalization
The most common error encountered in using HKPs as a loading control is overloading them.4 To accurately assess and normalize target protein levels, researchers must measure both the target and loading control proteins within the dynamic range of the assay. Generally, the target protein is present in significantly smaller quantities than the loading control protein. To measure these lower target protein levels, researchers load a high level of sample, which may saturate the HKP levels in the gel lane. Such saturation of HKP levels is known as overloading. In such cases, the housekeeping proteins are no longer within the linear dynamic range for immunodetection.1 Consequently, changes in target protein levels become unreliable. For instance, a major concern regarding the use of beta-actin as a housekeeping protein is that at target protein concentrations commonly used by researchers, the beta-actin optical density values are often found to be beyond the linear range and thereby, uncorrelated with protein concentrations.5 To prevent overloading, researchers should test that the housekeeping protein signal is linear and within the system’s dynamic range.
The second major concern in using HKPs as a normalization tool is the lack of any universal housekeeping protein. In fact, studies—similar to guidelines issued recently by the PCR scientific community6—often recommend that Western blotting results be normalized using multiple housekeeping proteins7 and that housekeeping proteins should be optimized for each application. The quantities of HKPs present are dependent on parameters such as tissue type, disease state, sample preparation method, environmental condition, and whether they are application tested. Optimizing HKPs is often overlooked or bypassed because extensive work is required to validate the use and stability of each HKP as a normalization tool, adding significant time and cost to experiments.8 Each HKP should be individually optimized with respect to antibody dilutions, experimental conditions, incubation times, and image settings7 and validated for each application under the experimental conditions.
In recent years, alternative methods have been developed that more reliably and efficiently normalize Western blot results, improving assay performance while saving time and money.
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.
The majority of researchers who perform Western blots to quantify relative protein expression levels use housekeeping proteins to correct for errors associated with sample loading and gel transfer. However, scientists must exercise great care to avert either using the wrong HKP or overloading HKPs. Total protein normalization methods, either with or without staining, overcome the issues with HKPs and help achieve more reproducible results at faster speeds.11 With stain-free technology and other high quality total protein stains, western blots can be a perfectly reliable quantitative tool.
Aldrin V. Gomes, Ph.D. (email@example.com), currently serves as an editor of several journals including the Journal of Molecular and Cellular Cardiology and is an Assistant Professor in the department of Neurobiology, Physiology, and Behavior at the University of California, Davis. Ning Liu, Ph.D. (firstname.lastname@example.org), joined Bio-Rad as an application scientist and now is a product manager in the Laboratory Separation Division.
1 Taylor, S. C., Berkelman, T., Yadav, G. & Hammond, M. A Defined Methodology for Reliable Quantification of Western Blot Data. Mol. Biotechnol. 5, 217-26 (2013).
2 Sawa A, Khan AA, Hester LD, Snyder SH. Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death. Proc Natl Acad Sci U S A. 94, 11669-74 (1997).
3 Liu NK, Xu XM. Beta-tubulin is a more suitable internal control than beta-actin in western blot analysis of spinal cord tissues after traumatic injury. J Neurotrauma. 23,1794-801 (2006).
4 Gilda, J. E. & Gomes, A. V. Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Anal. Biochem. 440, 186-188 (2013).
5 Aldridge, G. M., Podrebarac, D. M., Greenough, W. T. & Weiler, I. J. The use of total protein stains as loading controls: an alternative to high-abundance single protein controls in semi-quantitative immunoblotting. J. Neurosci. Methods 172, 250-254 (2008).
6 Bustin, S.A. et. al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem. 55:4, 611-622 (2009).
7 Gürtler, A. et al. Stain-Free technology as a normalization tool in Western blot analysis. Anal. Biochem. 433, 105-111 (2013).
8 Colella, A. D. et al. Comparison of Stain-Free gels with traditional immunoblot loading control methodology. Anal. Biochem. 430, 108-110 (2012). Biochem. S
9 Hammond, M., Kohn, J. E., Oh, K., Piatti, P. & Liu, N. A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation. Bio-Rad Bulletin 6360 (2013).
10 Traditional vs. V3 Western Workflow at <http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6356.pdf>
11 Maintain the integrity of your Western Blots at <http://info.bio-rad.com/ww-v3-western-workflow-lp-f1.html>