for western blot - (Sep/07/2007 )
hi, guys, i am doing western blot and have some questions
1)the blocking buffer we use is odyssey, is every blocking buffer the same function? or different protein use different blocking buffer?
2)we use Tween-20 as detergent, but we store them in room temperature,would then think it might make it unstable?
3)when adding secondary antibody, we add SDS also, why?
4)why we keep blot in dark after adding secondary antibody?
thanks!
-batman2003-
QUOTE (batman2003 @ Sep 7 2007, 12:08 AM)
hi, guys, i am doing western blot and have some questions
1)the blocking buffer we use is odyssey, is every blocking buffer the same function? or different protein use different blocking buffer?
2)we use Tween-20 as detergent, but we store them in room temperature,would then think it might make it unstable?
3)when adding secondary antibody, we add SDS also, why?
4)why we keep blot in dark after adding secondary antibody?
thanks!
1)the blocking buffer we use is odyssey, is every blocking buffer the same function? or different protein use different blocking buffer?
2)we use Tween-20 as detergent, but we store them in room temperature,would then think it might make it unstable?
3)when adding secondary antibody, we add SDS also, why?
4)why we keep blot in dark after adding secondary antibody?
thanks!
q1: depends more on the antibody than the antigen
q2: stored as stock solution or in solution; as stock solution it is stable, in solution it is also stable but become contaminated if not sterilized
q3: it is not a general protocol
q4: it is not a general protocol or you mean developing with ECL? then you need dark to detect the chemiluminescence
-The Bearer-