Follow these tips for improving the reproducibility of your western blot assay, and for achieving experimental results that are actually publishable.
Each antibody-antigen interaction has unique characteristics. The interaction of antisera with protein epitopes in western blotting depend upon a number of factors, all contributing to the final signal/noise ratio. Gaining knowledge and control of those factors allows for modifications and optimization of the procedure.
To ensure that the target protein is in your starting material, it is essential to prepare and store your samples to prevent degradation of protein and formation of aggregate. If the target protein is degraded or not present in sufficient amounts in the loaded sample, it can result in a lack of signal detection. Epitope abundance can be enhanced under altered growth conditions, by selective tissue and cell preparation, or fractionations of complex cellular extracts (i.e. organelle preparation). Use the extraction method which provides good yields and consistent results. For work with organisms which possess robust cell walls, such as bacteria or plant cells, you should use intensive homogenization and an appropriate inhibitors cocktail.
Clean, clean, always clean…
Other practices we recommend you follow to achieve reproducible results include use of gloves, forceps, clean and detergent-free laboratory materials, reagents which have not expired, and freshly prepared buffers with a controlled pH value.
Additionally, use of an automated system for processing, such as BlotCycler, is extremely helpful for closely following assay protocols, maintaining uniform volumes and dilutions, and precisely controlling incubation times. You can improve western blot reproducibility.