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Boiling a Western Blot? - Does it work? (Oct/16/2008 )

I have read on this forum that boiling a western blot after the transfer for 10 min helps to fix the protein to the membrane.

Does anyone have experience with this? How/why does this work?

Will it work on PVDF and Nitrocellulose? Phosphorylated proteins? Membrane bound proteins?



Oh dear, I would not try to boil a western membrane when you want to detect phosphorylated proteins! I think this is really not necessary. Your proteins are fixed on the membrane by the blotting procedure itself and will not go away (only smallest portion, if at all, after stripping). You can strip a PVDF membran for 3-5 times and still detect proteins.


Yes that was my original reaction.


QUOTE (strong32 @ Oct 16 2008, 08:14 AM)
Yes that was my original reaction.

where did you hear that exactly on this forum?

PVDF and nitrocellulose membranes are very sensitive/fragile...I wouldn't do that to be honest.

as the previous poster said you can strip your antibodies 3-5 times with stripping buffer and still proteins are there.