1 reply to this topic

[kadamb]

I am doing total protein extraction from cell lines and most of the time after extraction of protein, i get pellet which contains lots of protein which i dissolved in SDS sample buffer. Running normal gel shows many protein bands which i want to seperate by IEF but i am not sure how much % SDS, IEF can tolerate i have read in one of the paper but couldnot find. Any idea all suggestions are welcome.
Thanks a lot.
kadamb

(Edited by kadamb at 1:55 am on June 7, 2001)

[Mike]

I routinely use SDS in denaturing buffer (DB) for IEF:
3X DB: SDS 10%, DTT 150 mM. Aliquot and store  at -80C.
IEF Sample buffer (ISB): DTT (65 mM), CHAPS (65 mM), Urea 9 M, ampholytes 2%, filter sterilize, store -80C.
Mix your sample with 1/2 vol of DB, boil 1 min then mix with 2-4 vol of the ISB.