I am working with a protein located in the mitochondrial inner membrane, and I would like to know in which conditions should I perform the denaturation step... maybe the "standard" denaturation at 95ºC-100ºC for 5 minutes does not work for that kind of proteins.
Thank you very much.
Dear Marta García López
to my knowledge with membrane proteins is better to avoid the standard thermal denaturation at 95°C since the high temperature can induce protein aggregation but it is better or avoid thermal denaturation or preform a weaker incubation (3g- 55-60°C)
https://igakkai.kms-igakkai.com/wp/wp-content/uploads/2021en/KMJ-E202147013.pdf
Article Transmembrane protein western blotting: Impact of sample pre...
good luck
Manuele
Avoid to heat that much, it may lead to aggregate formation. In general, incubation in Laemmli buffer (containg SDS of course) at 37°C for one hour or so must be enough to give correct results on migration and Western Blot.
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