Protein gels are an invaluable tool for many researchers, offering an easy and relatively inexpensive way to compare protein expression between samples or to see how clean a protein is during a purification. Coupled with western blotting, they provide positive identification of protein expression in an easily interpretable format, making them one of the most widely used methods to validate antibody specificity. For both protein gels and western blots, consistency is critical for good results. Discussions in recent years have focused mainly on methods to improve western blot reproducibility, resulting in the evolution of specialized imagers for western blot analysis free of user-bias. To reap the benefits of these systems, it is important to remember that proper upstream processing remains essential to data quality.
Many small changes add up to noticeable variability
Although relatively simple to perform, western blotting involves numerous steps, each of which can introduce variability. In combination, even tiny changes can lead to inconsistent results. Lisa Isailovic, VP of marketing at Azure Biosystems, explains that since biological variability remains largely outside of researchers’ influence, it is important to keep the variables that can be controlled to a minimum. “The cells or tissues you’re collecting samples from should be handled as similarly as possible,” she says. “This includes how they are grown, as well as how they are processed into lysate. If you want to compare multiple gels or blots, these must be run using identical conditions every time, and should share a single control sample. Minimizing variability will help to ensure data quality.”
Total protein or housekeeping protein?
The most appropriate method to normalize western blot data is an ongoing debate. Jeff Harford, senior product marketing manager at LI-COR Biosciences, notes that best practices from publishers such as the Journal of Biological Chemistry generally recommend total protein staining to reduce the variability and error that can be introduced by normalizing data with a single housekeeping protein. This can be achieved using stain-free technology or with a total protein stain such as LI-COR’s Revert™ 700. “A key benefit of total protein staining is that it allows you to monitor protein transfer across the entire membrane at all molecular weights,” says Harford. “That said, the decision to use a total protein stain will depend on the nature of the work being performed. For example, researchers studying PTMs often use the pan-form of the target as the loading control.”
“If researchers choose to use a housekeeping protein to normalize data, they should select a protein demonstrating consistent expression,” notes Sam Heaton, associate scientist at Abcam. “A good example is GAPDH, which catalyzes the sixth step of glycolysis and as such is expressed in the majority of cells and tissues. However, it is essential to confirm that the expression of any housekeeping protein is not impacted by experimental conditions.” A further consideration for quantitative western blotting is to ensure the loading control and the target are detected within the same linear range, since otherwise the relationship between the two proteins will be largely unknown.
Well-validated antibody reagents are vital
Antibody reproducibility remains a hot topic, applicable to any immunostaining technique, with antibody specificity being a major factor in the usefulness of western blot data. Offering better experimental reproducibility over traditional antibody formats, and the opportunity to improve specificity and sensitivity through antibody engineering, recombinant monoclonals are becoming the preferred format for many researchers. “To ensure that western blot results are correct, it is advisable to choose extensively validated antibodies,” reports Heaton. “These allow for accurate comparison of multiple related samples without introducing concern that observed effects are due to antibody performance. To assure confidence in experimental data, we knockout validate many of our recombinant monoclonal antibody products, testing them in key applications including western blot.”
Are dark rooms becoming obsolete?
The use of gel documentation systems is a highly popular approach to reduce variability. “Using film to develop blots is not quantitative and really should only be used for confirming the presence or absence of a signal,” explains Lindsey Kirby, product manager at Syngene. “Multiple film exposures from the same blot will inevitably be captured under differing conditions, meaning that the data you depend on to make accurate comparisons are constantly changing. By switching from film to a gel documentation system such as one of our Syngene imagers, researchers can capture both faint and bright bands on the same blot without over-saturation. Moreover, gel documentation systems save an electronic copy of the image, making it much easier to store and share data.”
Heaton notes that modern gel documentation systems often allow for imaging in several channels, including both chemiluminescence and fluorescence, providing a significant improvement on film-based methods. Supported by powerful software designed for reproducible image analysis, these reduce user-bias by minimizing the level of input required to capture and quantify western blot data. LI-COR’s Empiria Studio™ Software, Azure Biosystems’ AzureSpot, and Syngene’s GeneTools software all incorporate best practices directly into western blot analysis, resulting in data with improved accuracy.
Image: AzureSpot software from Azure Biosystems benefits from features including automatic detection of lanes and bands, correction for background, and calculation of molecular weights to improve the reproducibility of western blot data analysis.
Protein gels are here to stay
With their relevance to so many applications, not least western blotting, protein gels remain a trusted tool to support scientific research. “Technology has come a long way, and with the latest knowledge and best practices, protein gels can be a fantastic tool for understanding protein expression,” says Harford. “But it is important that researchers take some time to appreciate the differences today from traditional methods to take full advantage of the many benefits it can offer.”
Minute™ Spin – improving data quality through efficient total protein extraction
Using traditional solution-based methods of protein extraction such as RIPA buffer, researchers are unwittingly introducing artefacts into protein quantitation data. Relying on weak lysis agents to address the stickiness of genomic DNA, and chemicals to prevent DNA unfolding, RIPA buffer delivers slow, inefficient protein extraction and non-selective, non-systematic protein loss. This equates to an unwanted ratio change for a given protein against the entire protein population and leads to questionable data interpretation, especially in quantitative studies dependent on band intensity in PAGE.
Minute™ Spin from Invent Biotechnologies helps achieve a complete protein profile after protein extraction, according to Quanzhi Li, CTO of Invent Biotechnologies. Based on a simple spin-column methodology, Minute™ Spin extracts proteins from cells or tissue samples in as little as 1 minute and can be used with small sample sizes from any living organism or tissue type to produce an unaltered endogenous baseline. “Suitable for protein trafficking/translocation, protein-protein interaction, protein modification, and signal transduction studies, as well as daily protein analysis, Minute™ Spin is a simpler, smarter approach to total protein extraction that improves data quality,” he adds.