PART 2 OF A 3-PART SERIES ON RELIABLY QUANTIFYING WESTERN BLOTS

Editor’s note: In part one, you learned about some of the hurdles to successful Western blotting and how to gain more confidence in your results using a “trust, but verify” approach. In this segment, we’ll discuss one of the most often used—and abused—tools to quantify Western blots: housekeeping proteins.

Researchers are always seeking to hedge against technical errors and inconsistencies that can put their data at risk. With Western blotting, such errors often arise during sample preparation, loading and transfer steps. Variations in transfer efficiency, for instance, can result in a two- to four-fold increase or decrease in the signal between gel lanes,¹ and that can hinder results.

Traditionally, researchers have turned to loading controls—most commonly, housekeeping proteins such as GAPDH, beta-actin and tubulin—to prevent these technical errors. Because these proteins are constitutively expressed and maintain cell viability, they are thought to accumulate at constant levels under all conditions and in all cell types. Normalization becomes even more important when measuring very small differences in protein expression between samples.

However, there are two major concerns associated with housekeeping proteins that jeopardize their validity as loading controls. To ensure the highest-quality data from your experiments, it’s necessary to avoid both.

Normalization With Less Risk

The first potential problem to overcome is overloading. To reliably assess changes in target protein levels, researchers must measure both the target and loading control proteins within the linear dynamic range of the assay. However, housekeeping proteins often end up being overloaded in the gel lane with the target protein.²

This happens because housekeeping proteins are also the most abundant proteins in a cell or tissue, whereas target proteins are often found in low abundance. Most labs typically have to load large amounts of cell lysate to detect the presence of their target proteins. Overloading results in such a high abundance of housekeeping proteins that they are out of the linear dynamic range for immunodetection. Therefore making any quantitative analysis based on this kind of data unreliable.

The second potential downfall of housekeeping proteins is that their expression can vary according to experimental conditions. Scientists typically use these proteins under the assumption that their expression levels remain consistent among different samples. Plenty of studies indicate that housekeeping protein expression changes under many scenarios, including siRNA treatment, cell death, and cell differentiation (See Bulletin 6350: Housekeeping Protein Publications²). Inconsistent expression levels render these proteins ineffective for the normalization of Western blots.

Scientists who understand these limitations validate their housekeeping proteins under the exact experimental conditions they plan to use. But even if properly validated, stripping and re-probing for housekeeping proteins adds several hours to the protocol, not to mention extra expense.

There is hope, however. New alternatives have emerged to make normalization more reliable and efficient for Western blots. For example, studies on HKPs have shown that total protein stains are better suited to correct for differences in loading than single-protein, high-abundance loading controls³. Researchers can simply stain the blots with widely available total protein stains such as Coomassie, Flamingo Pink, Sypro Ruby, Amido Black, or Ponceau S.

These stains allow for measurement of total protein signal within each lane as a loading control. Because total protein staining exhibits excellent linearity in measuring the protein load between 10-50 μg which is commonly used in western blotting, it eliminates the possibility of gel saturation, providing researchers with a true reflection of the amount of protein loaded in each lane.⁴

Stain-free Technology Worth Exploring

While total protein stains are free from the challenges associated with housekeeping proteins, they require time-consuming staining and destaining procedures. Staining may also introduce the possibility of experimental errors due to an affinity for binding to artifacts on membranes, resulting in an uneven background signal. Researchers at Bio-Rad have introduced an innovation in Western blotting: a stain-free technology that enables researchers to reap the benefits of total protein staining without any of the drawbacks. ^{5, 6, 7}

Stain-free technology uses a unique in-gel fluorescent compound that irreversibly binds to tryptophan residues, which allows researchers to directly visualize and quantify proteins both in gels and on blots. The procedure is simple, offers sensitivity comparable to conventional protein staining, and provides better reproducibility.

How much time does this technology really save? Proteins can be fluorescently visualized in the gel and analyzed in minutes. Furthermore, the fluorescence is maintained throughout the process so that the proteins can be imaged at any point after separation. [See Infographic: Traditional and V3 Western Workflow.⁸]

Using this technology, researchers can gain greater confidence in their data while reaching their end results faster.

Now that we’ve explored one of the biggest challenges for Western blotters, housekeeping proteins, we’ll move on to another major data killer: film. Continue on to the third part of this special series to learn more about how to make the most of your Western blots.

For more information on western blotting methods, ideal transfer conditions, and troubleshooting western blots, visit Western Blotting - Applications and Technologies.

References

1. Taylor SC, Berkelman T, Yadav G, Hammond M. A Defined Methodology for Reliable Quantification of Western Blot Data. Mol. Biotechnol. 2013. doi: 10.1007/s12033-013-9672-6. Published May 2013.

2. Aldridge GM, Podrebarac DM, Greenough WT, Weiler. The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J. Neurosci. Methods. 2008. 172(2): 250-254. doi: IJ10.1016/j.jneumeth.2008.05.003. Published July 30, 2008.

3. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6349.pdf

4. Li R, Shen Y. An old method facing a new challenge: Re-visiting housekeeping proteins as internal reference control for neuroscience research, Life Sci. 2013. 19(32): 747-751. doi: http://dx.doi.org/10.1016/j.lfs.2013.02.014. Published April 19, 2013.

5. Gilda JE, Gomes AV. Stain-Free total protein staining is a superior loading control to β-actin for Western blots. Anal. Biochem. 2013; 440(2): 186-188. doi: http://dx.doi.org/10.1016/j.ab.2013.05.027. Published September 15, 2013.

6. Gürtler A, Kunz N, Gomolka M et al., Stain-Free technology as a normalization tool in Western blot analysis, Anal. Biochem. 2012; 432(2): 105-111. doi: http://dx.doi.org/10.1016/j.ab.2012.10.010. Published February 15, 2013.

7. Colella AD, Chegenii, N, Tea MN, Gibbins IL, Williams KA, Chataway TK. Comparison of Stain-Free gels with traditional immunoblot loading control methodology, Anal. Biochem. 2012; 430(2): 108-110. doi: http://dx.doi.org/10.1016/j.ab.2012.08.015. Published November 15, 2012.

8. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6356.pdf