How to remove protein to show an activity is protein based - (Apr/04/2011 )

Hey,


So I would like to show that a specific activity is protein based. To do this I want to destroy the proteins in the extract and check if activity is gone. I cant heat denature as many of the stress related proteins that I work on are stable at high temperatures and the like. I was thinking of using an enzyme to do it, would proteinase K work?

Thanks

Proteinase K will destroy all the proteins in solution. How about knocking the RNA down with RNAi before making the lysate.

yeah, RNAi sounds better. Or maybe you can remove your protein from your lysate using an specific antibody

Destroying all the proteins will work fine, I just need to show that when the proteins are gone that the activity is gone, if the activity is still there then I know that there may be sugars in the lysate that are causing the activity ie that is not protein based.

What kind of concentration of proteinase K should definitely destroy all of them, I use it regularly to remove proteins in DNA extractions but would I need to up the concentration?

I would try heat first... It's easy to do and, if it eliminates the actvity, you can say the enzymatic activity is due to a heat labile protein. If it doesn't work, then you can try more difficult thinks like PK digestion, etc.

lorimc on Tue Apr 5 11:00:56 2011 said:

Oh will definitely be using heat but it has already been shown that heat had no effect on the activity of these proteins so I wanted to have the other method ready.

Thanks everyone

lorimc on Wed Apr 6 14:26:22 2011 said: