The Western blot (WB) is a commonly used technique for protein analysis. With this single assay, individual proteins can be assessed for molecular weight, protein modifications, relative abundance and protein localization and interactions. The key for a successful WB is to use an antibody that specifically reacts with the target protein without significant cross-reactivity. A successful blot also relies on a signal/noise ratio that allows detection of the protein with minimal background. High background is a very common problem in Western blots.
Many factors can contribute to high background in WB, such as improperly diluted antibodies, insufficient blocking, interference from blocking reagents, insufficient washing and poor quality of transfer membranes. However, the method/reagent used for protein extraction/sample preparation has been overlooked. Few researchers actually realize that the methods for protein extraction can significantly impact the background. The following two figures show side by side comparisons of the effect of protein extraction methods on WB background when RIPA buffer was compared with the spin column-based MinuteTM Protein Extraction Kits.