all intensities disappear after stripping - Western blot (Sep/08/2005 )

After I have stripped the filters then one of my filters lost all the signals even the signals of the MW marker. What is wrong? The protocol for stripping is this:


Warm the stripping buffer (62,5mM Tris-HCl pH 6.7 and 2% SDS) to 50C and put the filter in it for 30min at Room Temp (RT). Wash 2X 10min at RT. Block the membrane with 5% non fat dry milk solution.

Any ideas?

Thank you!

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maybe I misinterpreted your question, but isn't the aim of stripping a westernblot the complete loss of all residual signals, so that the same membrane can be re-used and re-probed with another antibody?

mike

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hi,
i hope u want to strip the membrane to perform reprobing. if this is your interest then obviously u should loose the signal after stripping, otherwise in next round of probing with antibodies will give u additional signals.
think of your problem, when u said that "one of your filters lost the signal while stipping" means did u use same antibody for all the membranes which u stripped. if not the antibodies which u used for this blot might be of less specific to your protein, so it easily got ditached when compared with other antibodies used for different filters.
if you used same antibodies for all the membrane which u stripped, then there must be some other reason, that could be a vriation in protocol or a variation in reagent(old or fresh) used for stripping.

my stipping protocol, is warm stipping buffer for 30 min/37C and incubate membranes in the buffer for 5 min, proceed with washings and blocking. if antobodiy is strong, increase the incubation time in stipping buffer.

with this comment i hope u would be in a better position to judge yourself.
plese make an arguement with me, if u find that my comment is not logical

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