probing for pAMPK:
blocking - 5% bsa
primary - 2% bsa o/n
secondary - 2% bsa
washings with 0.1% tbst
my queries are:
should i be blocking with nfdm?
and secondary also with nfdm?
and for total ampk
should continue with bsa?
or with milk for blocking /primary/secondary?
4% v/v Odyssey Blocking Buffer in TBST works well for all antibody incubations, particularly if you are detecting secondaries by a fluorescent method. But the other blocking agents mentioned (diluted in TBST) should also work. There should be no need to switch between blocking agents for primary and secondary antibody incubations.
Muhammad,
I use block and diluent for primary as suggested by the manufacturers. Cell Signalling is a popular p-AMPK AB and they say do not use milk, but BSA. I then switch to normal goat serum for my goat anti-primary fluorescent secondary(s). All include 0.1% Tween-20, as well as the TBS washes. It often ends up trial and error. Some AB's do not work well in milk. Some don't work well with BSA. The fish based non-mammalian block's are another thing to try when milk or BSA doesn't work.
Good luck.
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