: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
SS: Journal standards for publishing Western blot data are becoming more rigorous, but clear standards for antibody validation are not yet established. Validation results and experimental methodologies for Western blot analysis are not consistently reported in the literature, making it difficult to evaluate published results.
Careful experimental design is critical for quantitative Western blotting, but its impact on accuracy and reproducibility is not widely recognized. Sample preparation, detection, image capture, and data analysis methods can introduce variability and limit the reproducibility of Western blot analysis. For accurate quantitative analysis, it is important to understand and eliminate common sources of error. The key is to maximize accuracy and precision by minimizing Western blot variability, so your relative comparisons are as meaningful as possible. Careful validation and documentation of antibody performance are critical parts of this process.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
SS: LI-COR’s near-infrared (NIR) fluorescent secondary antibody products are affinity purified and highly cross-adsorbed to ensure high specificity and minimal cross-reactivity. All IRDye® antibody conjugates are specifically tested and qualified for fluorescent Western blotting. Secondary antibodies labeled with spectrally distinct IRDye fluorophores can be combined for multiplex Western blot detection.
When purchasing antibodies from a vendor, we choose primary antibodies that are specifically validated for Western blotting. For multiplex Westerns, careful selection of primary and secondary antibodies is critical to prevent cross-reactivity. We choose primary antibodies raised in two different host species, which can be discriminated by the secondary antibodies. Secondary antibodies must be highly cross-adsorbed, and should be derived from the same host species if possible. To validate antibodies for multiplex Western blotting, we run three control blots—a separate blot with each individual primary antibody, and a third blot with the primary antibodies combined. These controls will characterize the banding patterns and verify that the primary antibodies can be multiplexed.
: Do you have any advice, recommendations, and/or best practices scientists should follow when selecting and validating an antibody?
SS: The presence of a single band at the expected molecular weight on a Western blot is a great start but it is not sufficient for antibody validation. Combining several validation strategies is the most effective way to demonstrate antibody specificity and validate performance. For example, it is important to demonstrate antibody specificity and selectivity for endogenous levels of target protein expression; purified or overexpressed target protein should not be used for validation. You can incorporate knockdown or knockout strategies to decrease the abundance of your target, or treat cells with growth factors or chemical compounds that induce or inhibit expression of the target. A blocking peptide that contains the epitope recognized by your antibody can be used to prevent binding to the target protein. And you should always record the antibody name, source, catalog or clone number, lot number, host species, immunogen, and other information that may be needed later.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
SS: Reproducibility in scientific research is a complex problem and as scientists, we have a shared responsibility to identify, understand, and address the factors that contribute to this problem. The increased visibility and awareness of reproducibility issues generated by the publishers of Nature and the Journal of Biological Chemistry (JBC), the National Institutes of Health’s rigor and reproducibility initiative, and the 2016 GBSI workshop on antibody validation are huge steps in the right direction.
These efforts are realistic and badly needed, but transforming guidelines and recommendations into standards is not easy or straightforward. Standards must be flexible enough to adapt to the experimental context, without being overly complex or burdensome. Awareness and training are important aspects of implementation. Open communication of antibody performance and validation data, along with consistent reporting of detailed validation and experimental methodologies by authors, will also be critical for success.
: What else is your company doing to address reproducibility?
SS: LI-COR is leading the way to improve reproducibility for researchers that do Western blotting, an application that, of course, requires both validated primary and secondary antibodies. Following JBC’s lead in releasing new requirements to help researchers generate and submit more reproducible Western blot data, we have developed new methods and protocols to assist LI-COR Odyssey® users in successfully addressing these new requirements. We have found in our own studies that these new methods can help researchers generate more reliable, reproducible Western blot data.
New protocols for generating more reproducible, reliable Western blot data explain how to:
Author Bio: Steve Shiflett is a Technical Product Manager at LI-COR Biosciences. He received his M.S. in biochemistry and molecular genetics from the University of South Florida. Since 2000, he has spent most of his career developing protein biology and cellular imaging/analysis products.