What should I take into consideration before I strip and reprobe my western blotting experiment?

There are several things one should consider before striping and reprobing their western blot experiment. One thing to consider is to carry out a negative control. This involves testing the efficiency of the stripping protocol by adding a substrate to the stripped blot. If no signal is generated, it confirms the successful removal of previous antibody signals. However, if bands still appear, one should strip for a longer duration or use a more stringent stripping protocol. Another consideration is to maintain the moisture of the blot throughout the entire process. Drying out the membrane can lead to permanent binding of antibodies, and thus affect the results. One should also ensure that the detection method is suitable with stripping. Stripping is effective only with chemiluminescent or radioisotope detection systems. This is because colorimetric substrates such as BCIP, 4CN, TNB and DAB form a precipitate on the membrane that remains unaffected by antibody dissociation. Lastly, it's crucial to assess the impact of the stripping protocol on the targeted proteins. Failure to do so may limit the interpretation of results to qualitative analysis only. To optimize the stripping protocol, one should compare the signal strength of the blot before and after stripping using a specific antibody. A decrease in signal intensity suggests inadvertent removal of target proteins.