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 always 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
