We've got it to work fine from a purely "look and see" perspective, but
never applied it to anything yet, so don't know how it might work if you
took it to the next step - i.e. probing with Ab's. We used a "wet"
(Towbin) transfer, to nitrocellulose, 2hrs at 100V in the cold room. Very
little was left on the gel afterwards. Be sure to cut the blue "blob"
from the bottom of the gel first, as it makes a real mess otherwise. The
important thing is to equilibrate the gel for at least an hour with some of
your transfer buffer (include 0.05% SDS in it). Try rinsing a few times
too, to remove as much coomassie blue as possible. The object of the game
is to replace some of the coomassie with SDS, so that the proteins will
have a negative charge in the Tris-Glycine buffer system and will thereofre
migrate. The end result is a bit of a mess - a blue membrane with darker
bands on, just like the gel really.
Good Luck
PSB
_________________________________________
Dr. Paul S. Brookes. (brookes at uab.edu)
UAB Department of Pathology, G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915 Fax (001) 205 934 1775
http://peir.path.uab.edu/brookes
The quality of e-mails can go down as well as up
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