Disadvantages of membrane stripping and reprobing - I need your opinion ASAP (Sep/28/2006 )
There are hundreds of application notes listing all advantages of membrane stripping. But I really need to formulate some sentences why in some cases stripping isn't the best idea to normalise initial signal.
Please, help with your opinion ASAP!!!
stripping removes antibiotics but alters also the protein quantity on the membrane are they're not covalently fixed.
So it's regulary assumed that the stripping procedure it roughly uniform, but it may not.
Moreover stripping may remove "excess" of proteins. I've noticed that for heavy proteins, some are visible at first try but not if stripped.
uncomplete "stripping" may result in blocked epitopes by still bound antibodies which disturb when re-probed with another polyclonal antibody against the same antigen, so specific signal does not correspond with amount of antigen/epitopes;
moreover, rest of bound antibodies even after stripping may be detectable by secondary antibody, and also interfere with re-probing as unspecific signal