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
- - - - -

co-IP of chromatin binding protein


  • Please log in to reply
2 replies to this topic

#1 beenyg

beenyg

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 27 December 2009 - 10:06 AM

Hi,

I've been trying (too long now) to find the right conditions for HEK-293 cell lysis in preparation for co-IP of my nuclear protein. This protein binds chromatin, and so using a delicate 150mM NaCl + 0.5% NP40 bufer leaves most of it in the pellet. I was told to add DNaseI and MgCl2 to the buffer, which I did, bringing on total degradation of the protein, in spite of protease inhibitor cocktail and PMSF (I've tried three different samples of DNaseI). So my questions are:
1) Does anyone know of specific problems with DNaseI? Would MNase be better?
2) Is there any reason to try nuclear extraction rather than the whole cell extraction I'm using now?
3) Anyone else working on Co-IP of chromatin binding proteins that has any tip on the issue would be much appreciated...

many thanks,
Benny.

#2 signstem

signstem

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 08 January 2010 - 09:09 PM

Hi,

I've been trying (too long now) to find the right conditions for HEK-293 cell lysis in preparation for co-IP of my nuclear protein. This protein binds chromatin, and so using a delicate 150mM NaCl + 0.5% NP40 bufer leaves most of it in the pellet. I was told to add DNaseI and MgCl2 to the buffer, which I did, bringing on total degradation of the protein, in spite of protease inhibitor cocktail and PMSF (I've tried three different samples of DNaseI). So my questions are:
1) Does anyone know of specific problems with DNaseI? Would MNase be better?
2) Is there any reason to try nuclear extraction rather than the whole cell extraction I'm using now?
3) Anyone else working on Co-IP of chromatin binding proteins that has any tip on the issue would be much appreciated...

many thanks,
Benny.



I'm facing a similar problem as you. To me it seems the nuclear envelope has been lysed; just that the protein binds to DNA too tightly. I read it somewhere that a higher NaCl concentration (say 500mM) may disrupt the protein-DNA association. Not sure whether it works or not, but I'm going to try. Just for your info.
Signstem

#3 vered

vered

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 22 June 2010 - 01:48 PM

i have the same problem and i just posted a Q in the forum. if through my tries ill come up with something ill sure let you know.
V

Hi,

I've been trying (too long now) to find the right conditions for HEK-293 cell lysis in preparation for co-IP of my nuclear protein. This protein binds chromatin, and so using a delicate 150mM NaCl + 0.5% NP40 bufer leaves most of it in the pellet. I was told to add DNaseI and MgCl2 to the buffer, which I did, bringing on total degradation of the protein, in spite of protease inhibitor cocktail and PMSF (I've tried three different samples of DNaseI). So my questions are:
1) Does anyone know of specific problems with DNaseI? Would MNase be better?
2) Is there any reason to try nuclear extraction rather than the whole cell extraction I'm using now?
3) Anyone else working on Co-IP of chromatin binding proteins that has any tip on the issue would be much appreciated...

many thanks,
Benny.



I'm facing a similar problem as you. To me it seems the nuclear envelope has been lysed; just that the protein binds to DNA too tightly. I read it somewhere that a higher NaCl concentration (say 500mM) may disrupt the protein-DNA association. Not sure whether it works or not, but I'm going to try. Just for your info.
Signstem






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

©1999-2013 Protocol Online, All rights reserved.