Western blot processing is a well-established procedure that includes protein extraction from tissues and cells, gel electrophoresis separation, transfer to a membrane, and immunodetection with specific antibodies. Optimization of washing helps to maximize the specific interactions of antigens and antibodies. Performing all washing steps at 4 degrees C ensures a maximal signal to noise ratio and reduces nonspecific signals. The interaction of Ab with the target is practically irreversible at 4 degrees C, however at room temperature there is significant dissociation. Based on our and our customer’s observations it is optimal to do all western blotting steps at 4 degrees C; this will result in higher signal, less background and helps to eliminate nonspecific bands. Since each primary antibody is unique, protocol optimization can be challenging. Washing and hybridization require consistent agitation and precise timing. Both these steps are difficult and not practical using a manual procedure; no one can be expected to run to the cold room every time to change washing buffer. If the goal is to produce consistent and reproducible western blotting results it is important to implement an automated blot processing system. Hands free western blot processing using either the Precision Biosystems BlotCycler Touch or BlotCycler Mini enables researchers to optimize all aspects of the blot processing cycle. Automating, blocking shaking, washing, and antibody incubation eliminates nonspecific bands and generates high quality reproducible results.
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