co-IP of chromatin binding protein - (Dec/27/2009 )
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.
beenyg on Dec 28 2009, 02:06 AM said:
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
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
signstem on Jan 8 2010, 10:09 PM said:
beenyg on Dec 28 2009, 02:06 AM said:
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