A trouble with Immunoprecipitation - (Sep/24/2006 )
Hi people,
I'm trying to IPing a membrane transporter in platelets, doing this i've found that my protein migrate @ 50 kDa where the predicted mass is about 80 kDa. We think this is due to proteolysis, and we are unable to block this process using Complete mini anti-protease cocktail. The only condition that block proteolysis and make us see the protein migrate at 80 kDa is using Urea 8M in lysis buffer (we have confirmed that with WB exp). The problem is : Can i trying to IPing the transporter in a 8M Urea buffer? (i think not). Some1 of us know a way to eliminate Urea be4 IPing? (hoping this not activate proteolysis).
Thanks all
-alepd-
QUOTE (alepd @ Sep 24 2006, 11:17 AM)
Hi people,
I'm trying to IPing a membrane transporter in platelets, doing this i've found that my protein migrate @ 50 kDa where the predicted mass is about 80 kDa. We think this is due to proteolysis, and we are unable to block this process using Complete mini anti-protease cocktail. The only condition that block proteolysis and make us see the protein migrate at 80 kDa is using Urea 8M in lysis buffer (we have confirmed that with WB exp). The problem is : Can i trying to IPing the transporter in a 8M Urea buffer? (i think not). Some1 of us know a way to eliminate Urea be4 IPing? (hoping this not activate proteolysis).
Thanks all
I'm trying to IPing a membrane transporter in platelets, doing this i've found that my protein migrate @ 50 kDa where the predicted mass is about 80 kDa. We think this is due to proteolysis, and we are unable to block this process using Complete mini anti-protease cocktail. The only condition that block proteolysis and make us see the protein migrate at 80 kDa is using Urea 8M in lysis buffer (we have confirmed that with WB exp). The problem is : Can i trying to IPing the transporter in a 8M Urea buffer? (i think not). Some1 of us know a way to eliminate Urea be4 IPing? (hoping this not activate proteolysis).
Thanks all
Q1: should fail
Q2: differential dialysis, for small amounts try Pierce Slide-A-Lyzer, or tube/cup filtration methods
-The Bearer-