Dual cross-linking thoughts - Agents to help analyse proteins indirectly bound to DNA (Jan/21/2010 )
I routinely do ChIP assays for proteins not directly bound to DNA. I've compared DTBP pre-treatment to just formaldehyde fixation and found no difference in the efficiency of the ChIP. One thing that did make a big difference though was the cell lysis buffer. Try using a less stringent lysis buffer (i.e. with a lower SDS concentration than the standard 1%).
-annabellak-
annabellak on Mar 8 2010, 02:56 PM said:
I routinely do ChIP assays for proteins not directly bound to DNA. I've compared DTBP pre-treatment to just formaldehyde fixation and found no difference in the efficiency of the ChIP. One thing that did make a big difference though was the cell lysis buffer. Try using a less stringent lysis buffer (i.e. with a lower SDS concentration than the standard 1%).
Why lowering the SDS concentration can improve the efficiency of the ChIP? create less bubbles during sonication step? I used Upstate ChIP kit.
I tried DTBP before. It will enrich the DNA to which the protein binds directly but not the one which binds indirectly.
-giny-
giny on Apr 1 2010, 04:08 AM said:
annabellak on Mar 8 2010, 02:56 PM said:
I routinely do ChIP assays for proteins not directly bound to DNA. I've compared DTBP pre-treatment to just formaldehyde fixation and found no difference in the efficiency of the ChIP. One thing that did make a big difference though was the cell lysis buffer. Try using a less stringent lysis buffer (i.e. with a lower SDS concentration than the standard 1%).
Why lowering the SDS concentration can improve the efficiency of the ChIP? create less bubbles during sonication step? I used Upstate ChIP kit.
I tried DTBP before. It will enrich the DNA to which the protein binds directly but not the one which binds indirectly.
If the protein you're ChIPing is not efficiently crosslinked to the chromatin (e.g. not directly bound to the DNA or makes little contact with the DNA) then you are dependent on the protein's structure to maintain protein-protein or protein-DNA contacts. My guess is that lowering SDS concentration allows the proteins to maintain a more native state after sonication and thus maintain their contact with the chromatin.
-KPDE-
annabellak on Mon Mar 8 22:56:53 2010 said:
I routinely do ChIP assays for proteins not directly bound to DNA. I've compared DTBP pre-treatment to just formaldehyde fixation and found no difference in the efficiency of the ChIP. One thing that did make a big difference though was the cell lysis buffer. Try using a less stringent lysis buffer (i.e. with a lower SDS concentration than the standard 1%).
I've tried tinkering with the cell lysis buffer but all I got was a lot more background. But I know a lot of people that are able to improve their chip just by changing the SDS concentration...
-Radish-
so, Radish... what 5 regagents did you choose and why?, and is it possible to western the IPs??
-kika-
Radish on Fri Feb 26 22:19:48 2010 said:
Radish,
I am not using agent mentioned above. I overexpressed the proteins and I got the results! I would like to try using the agent now...could you please let me know the protocol? Thanks
For the question you asked, I have no idea whether we can western blot xlink samples. what is your approach? You harvest the samples, do IPs follow by western?
Hey Giny
I use the upstate protocol, so the only thing I've changed was the crosslinking.
I used attached cells (arround 2x107 cells in a 100mm plate) washed them with PBS+protease inhibitors 3 times, added the agents with 10 ml of PBS=PI (the agents were all solubilized in DMSO in 1 M stocks and added to the final concentration of 1,5 mM) incubated for 1 hour with agitation at room temp, washed again 3 times with PBS, added 10 ml of PBS+PI and 1 % Fa and incubated for 10 minutes. The rest of the protocol is the same, add the glycine yada yada.