Rf: To boil or not to boil?

[LOGAND]

>Dear netters.I have been doing western blotting for a year.
>I have not been boiling my tissue samples before
>loading. I am not adding b-mercaptoethanol either.
>I need to know the pros and cons of these two procedures.

Denaturation by boiling is known to cause aggreagation of certain 
polypeptides, particularly those which are sig. hydrophobic. Also it can 
cause problems for immuno-detection. Working with membrane proteins I do 
not boil (and have noticed the difference, ie. tested boil vs. 45C, with a 
putative Ca-ATPase and know that the H+-ATPase is sensitive to boiling 
(both plant enzymes). So ideally I would test boiling versus 45C. As for 
2-mercapto treatment, well I should think that will depend on whether you 
want your proteins reduced!

David C. Logan
INRA-ENSA-(M)-CNRS
Montpellier
FRANCE

Logand at msdos.ensam.inra.fr