7 replies to this topic

[lorimc]

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  

[bob1]

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

[laurequillo]

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

[lorimc]

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?

[HomeBrew]

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.

[newborn]

lorimc, on 05 April 2011 - 03:00 AM, said:

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 think you can use the following concentration for your purpose:
http://www.ncbi.nlm....pubmed/17336356

Using two method to prove your hypothesis is better as the previous blogger pointed out. You can use heating at the first step.

[lorimc]

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

[newborn]

lorimc, on 06 April 2011 - 06:26 AM, 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

No, I think you can use the conc of proteinase K for prion as the paper I suggested.