How many nitrocellulose blots can be safely stripped and incubated in primary/se - (Aug/21/2014 )
Today I stripped 6 nitrocellulose blots using a DDT mixture in the oven. I proceeded to probe for tubulin. When I went to develop the blots, I noticed that some entire blots had robust signals across the board, other membranes had faint signals throughout.
Is it likely that this is because all 6 blots were stripped and incubated in a single container? How does stripping and incubating multiple in a single container generally effect results? I have done this same procedure for 4 blots and had robust signals for every blot. Any and all advice appreciated - please share your experiences with stripping or incubating multiple blots at one time.
The problem with stripping is that it can be inconsistent - if one area of the blot is not convered by the stripping solution fully, then it won't strip properly and you will get inconsistent results. Personally I would say avoid stripping if at all possible, so if you proteins have 5 kDa or more difference in size, you should be able to differentiate with your different antibodies without stripping.