non-reducing IP - (Jan/26/2007 )
I am trying to detect whether dimerization/oligomerization of my target protein is required for sumoylation. Planning to run a non-reducing IP. Question is: Can I just boil the immunoprecipitated beads in presence of sample buffer (does it need to be SDS free) without any reducing agent (BME/DTT)? Will I expect the IgG band at ~150 kd? How about crosslinking the lysate before IP?
Any comment from people in similar situation will be appreciated.
-distantshore-
QUOTE (distantshore @ Jan 26 2007, 05:31 PM)
I am trying to detect whether dimerization/oligomerization of my target protein is required for sumoylation. Planning to run a non-reducing IP. Question is: Can I just boil the immunoprecipitated beads in presence of sample buffer (does it need to be SDS free) without any reducing agent (BME/DTT)? Will I expect the IgG band at ~150 kd? How about crosslinking the lysate before IP?
Any comment from people in similar situation will be appreciated.
Any comment from people in similar situation will be appreciated.
what you plan is native page; do not use SDS or reducing agents; non-denaturizing detergents such as cholate may help; most important is the buffer system;
may ask mdfenko for more details...
-The Bearer-