LI-COR
Technical Product Manager
For some researchers, optimizing simply means getting an experiment to work in the shortest amount of time for the sake of publishing. Yet the bedrock of science is reproducibility – a finding is useless if others cannot replicate it. The Reproducibility Initiative, which was awarded $1.3 million by the Laura and John Arnold Foundation to replicate the results for 50 of the most impactful oncology papers, underscores the importance of this principle.
At LI-COR, one of the companies sponsoring the Reproducibility Initiative, we have considerable experience (PDF) dealing with reproducibility in Western blotting experiments. But customers often struggle. Whether through lack of optimization or the use of improper controls, researchers often find it difficult to replicate results using Western techniques. To help, we offer the following tips for greater reproducibility in fluorescent protein blotting experiments.
1. Optimize membrane selection
To begin with, always minimize autofluorescence. A low-background membrane is essential when working with near infrared (NIR) wavelengths (600 nm to 700 nm)—like those of LI-COR’s Odyssey infrared imaging systems—and a specific membrane should be imaged out of the box to determine its autofluorescence. PVDF performance typically varies more than that of nitrocellulose membranes. Remember to use blunt forceps to handle membrane edges, as any scratching can create artifacts when imaging. Exposing the membrane to detergent before blocking is complete can also cause high background.
2. Optimize blocking solution
Traditional blocking solutions may be problematic, as milk-based blockers often contain lgG. IgG is notorious for cross-reacting with anti-goat antibodies, which can increase background and reduce sensitivity. Milk-based blockers may contain endogenous biotin or phosphorylated epitopes, too, which also can contribute to background. Remember, no universal blocker is best for all antibodies, so experiment accordingly. Particulates in buffers can settle on membranes and create fluorescent artifacts, so it’s worthwhile to use high-quality reagents and filter-sterilize all buffers.
3. Optimize primary antibody
Different primary antibodies for the same antigen can perform very differently. Therefore, test multiple primaries to determine the most suitable reagent. After you’ve identified your primary antibody, optimize its concentration to get the best results. One of main benefits of the Odyssey Infrared systems is the ability to multiplex two targets. Before combining primary antibodies in a two-color experiment, always perform preliminary blots with each primary antibody alone to determine the expected banding pattern and possible background signals. Always use highly cross-adsorbed secondary antibodies for two-color detection. Failure to do so can result in cross-reactivity.
4. Carefully consider loading controls
Most Western blot normalization (PDF) is performed by probing the membrane with antibodies against a housekeeping protein, because these are still widely assumed to express consistently across all types of tissues and experimental conditions. However, variability does occur—not only across cell types [1] but also cell treatments and environmental changes [2].When selecting a normalization standard, consider whether your experiment investigates: a) comparisons between different types of tissues, b) comparisons between treated and untreated cells within the same cell line or c) comparisons between normal and diseased tissue of the same type. Commonly used housekeeping genes—like GAPDH or β-actin—may be unsuitable for any one of these scenarios because of the influence of various physiological and pathological factors on their expression, especially in studies examining gene-transcript levels.
5. Validate normalization controls
Absolutely run an initial experiment under the same conditions as your final protocol to ensure that the expression of your normalization standard is not affected by factors like those mentioned above. Failure to do so may confound analyses downstream and thus reproducibility. Define the assay scope as well. Be certain the detection limits and linear range of detection for the normalization standard fall within the same parameters as your target. If your overall signal is too low, you may want to try increasing your protein load at the gel stage. There could also be an incomplete transfer of the proteins to blot or an antibody-concentration issue.
Conclusion
The idea that the same experiments always get the same results, no matter who performs them, is one of the cornerstones of science’s claim to objective truth. Reproducibility is the foundation of this premise. The Western blotting optimizations described above will help generate protein data that meet that criterion. As for the Reproducibility Initiative, a full disclosure of experimental results, many of which used Western blotting, is expected soon. Stay tuned.
References
[1] Tan, SC, et al., “Identification of valid housekeeping genes for quantitative RT-PCR analysis of cardiosphere-derived cells preconditioned under hypoxia or with prolyl-4-hydroxylase inhibitors,” Mol Biol Rep, 39:4857-67, 2011. [PubMed ID: 22065248]
[2] Banda, M, et al., “Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR,” Mutation Res, 649:126-34, 2008. [PubMed ID: 17904413]
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