High protease activity in protein extraction - A problem in western blot (Apr/29/2010 )
Dear all,
We encountered a problem that extremely low protein content is caused by very high protease activity when preparing the lysates from one of our marine animal for western blot and 2DE. The extraction protocol always works well and only this animal gave such high protease activity but not others. Actually, we added protease inhbitor cocktail and PMSF to prevent the activity of protease during lyzing, however, it seems far less than adequate to prevent the protease in this animal. So I just seek for help here. Thanks!
Kenny
-KXHK-
KXHK on Apr 30 2010, 03:39 AM said:
Dear all,
We encountered a problem that extremely low protein content is caused by very high protease activity when preparing the lysates from one of our marine animal for western blot and 2DE. The extraction protocol always works well and only this animal gave such high protease activity but not others. Actually, we added protease inhbitor cocktail and PMSF to prevent the activity of protease during lyzing, however, it seems far less than adequate to prevent the protease in this animal. So I just seek for help here. Thanks!
Kenny
We encountered a problem that extremely low protein content is caused by very high protease activity when preparing the lysates from one of our marine animal for western blot and 2DE. The extraction protocol always works well and only this animal gave such high protease activity but not others. Actually, we added protease inhbitor cocktail and PMSF to prevent the activity of protease during lyzing, however, it seems far less than adequate to prevent the protease in this animal. So I just seek for help here. Thanks!
Kenny
improve anti-proteolysis by working on ice, and be quick; also use protease inhibitors in sample buffers; shorten the time of electrophoresis running as well as soaking of IEF strips if possible
-Inmost sun-
Inmost sun on May 2 2010, 04:25 AM said:
KXHK on Apr 30 2010, 03:39 AM said:
Dear all,
We encountered a problem that extremely low protein content is caused by very high protease activity when preparing the lysates from one of our marine animal for western blot and 2DE. The extraction protocol always works well and only this animal gave such high protease activity but not others. Actually, we added protease inhbitor cocktail and PMSF to prevent the activity of protease during lyzing, however, it seems far less than adequate to prevent the protease in this animal. So I just seek for help here. Thanks!
Kenny
We encountered a problem that extremely low protein content is caused by very high protease activity when preparing the lysates from one of our marine animal for western blot and 2DE. The extraction protocol always works well and only this animal gave such high protease activity but not others. Actually, we added protease inhbitor cocktail and PMSF to prevent the activity of protease during lyzing, however, it seems far less than adequate to prevent the protease in this animal. So I just seek for help here. Thanks!
Kenny
improve anti-proteolysis by working on ice, and be quick; also use protease inhibitors in sample buffers; shorten the time of electrophoresis running as well as soaking of IEF strips if possible
Thnx. The protocol works well for other samples but seems not to work in respect to this species. We're trying to incorporate other protease inhibitors to see whether it could improve the protein content after lysis.
-KXHK-