Western blotting is an important technique used to identify specific proteins from a complex mixture of proteins extracted from cells. The technique uses three elements to accomplish this task: (1) separation by size, (2) transfer to a solid support, and (3) marking target protein using a proper primary and secondary antibody to visualize and quantify. Since each primary antibody is unique, protocol optimization can be challenging. Washing and hybridization require consistent agitation and precise timing; this can be accomplished using an automated system. It is important to perform at least one washing step after blocking to remove loosely attached blocking reagent, this can also help reduce background. A number of washing steps are then recommended to remove unbound antibody leaving only the bound antibody to the protein of interest. Consistently timed washing steps are often neglected due to manual manipulation. Based on our and our customers observations, washing at 4 degrees C produces higher signal and less background and helps to eliminate nonspecific bands. Hands free western blot processing using the BlotCycler Touch or the BlotCycler Mini enables researchers to optimize shaking, washing, and antibody incubation to eliminate nonspecific bands and generate high quality results.
