Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

How to remove protein to show an activity is protein based


  • Please log in to reply
7 replies to this topic

#1 lorimc

lorimc

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 04 April 2011 - 03:52 AM

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 :rolleyes:

#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,720 posts
399
Excellent

Posted 04 April 2011 - 03:56 PM

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

#3 laurequillo

laurequillo

    The Goddamn Batman!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 265 posts
1
Neutral

Posted 04 April 2011 - 08:26 PM

yeah, RNAi sounds better. Or maybe you can remove your protein from your lysate using an specific antibody
"He must be very ignorant for he answers every question he is asked" Voltaire

"This is SPARTA!"

"Im the goddamn batman"

#4 lorimc

lorimc

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 05 April 2011 - 03:00 AM

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?

#5 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
15
Good

Posted 05 April 2011 - 09:01 AM

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.

#6 newborn

newborn

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 71 posts
2
Neutral

Posted 05 April 2011 - 06:44 PM

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.

#7 lorimc

lorimc

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 06 April 2011 - 06:26 AM

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

#8 newborn

newborn

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 71 posts
2
Neutral

Posted 06 April 2011 - 04:27 PM

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.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.