14 replies to this topic

[Radish]

Hi everybody

Due to unsuccessful ChIPs I have been thinking about using a dual cross-linking protocol to my samples.
Apparently formaldehyde generates a limited spanning of just 2A, resulting in a short spacer arm, but, adding and extra reagent to the equation you can get a longer spacer arm, and this way reveal proteins that are more distantly bound to DNA (as for the case of transcription coactivators and corepressors).

I am trying to decide between the following:

-DSG (disuccinimidyl glutarate)
-EGS (ethylane glycol bis succinimidylsuccinate)
-DMA (dimethyl adipimidate dihydrochoride)
-DSS (suberic acid bis(N-hydroxysuccinimide ester)


Do you have any experience with any of them?


Thank you so much for your input guys.

[giny]

Hi Radish,

I tried imidoester crosslinker, DTBP. But after treatment, i lost quite a lot of cells as they will detach from the petri dish after treatment. And I do not see any DNA enrichment after treatment. So I stop using that.

Maybe you can try re-ChIP experiment. I have tried it but my boss prefer ChIP experiment follow by qPCR.

[Radish]

giny, on Jan 27 2010, 04:55 PM, said:

Hi Radish,

I tried imidoester crosslinker, DTBP. But after treatment, i lost quite a lot of cells as they will detach from the petri dish after treatment. And I do not see any DNA enrichment after treatment. So I stop using that.

Maybe you can try re-ChIP experiment. I have tried it but my boss prefer ChIP experiment follow by qPCR.

Hi giny

Well I had a detachment problem before so I just started to collect the cells after scraping and just proceeded with the crosslinking in a conical tube.

Right now I don't have a problem with my ChIP, I have a problem with a particular transcription factor that does not bind directly to DNA, I don't really have an alternative, I have to go for something with a longer spacer arm than formaldehyde.

The problem is that all those compounds are really expensive and controversial in there effectiveness at the same time.


Thank you for the input giny!

[giny]

Radish,

I have the same problem as you as I am analyzing a transcription factor that does not bind to DNA directly. I have tried many times already...still cant get the results. very frustrating with the ChIP.

If you found a solution to solve this, can you please let me know? I am wondering if ChIP can be used to analyze transcription factor tht doesnt bind to DNA directly.

thanks

[Radish]

giny, on Feb 4 2010, 03:01 AM, said:

Radish,

I have the same problem as you as I am analyzing a transcription factor that does not bind to DNA directly. I have tried many times already...still cant get the results. very frustrating with the ChIP.

If you found a solution to solve this, can you please let me know? I am wondering if ChIP can be used to analyze transcription factor tht doesnt bind to DNA directly.

thanks

Hello giny

Have you seen this paper?
In vivo dual cross-linking for identification of
indirect DNA-associated proteins by chromatin
immunoprecipitation


Any way, I am now trying a bunch of new conditions, hopefully I will have an answer by sunday.
I sure hope this works this time

[giny]

Hi Radish,

Thanks for the paper, I am reading now

Hope you will get the results this time round.

[giny]

Radish,

I got my results!!! Yeah, finally....and it's reproducible.

[Radish]

giny, on Feb 10 2010, 11:23 PM, said:

Radish,

I got my results!!! Yeah, finally....and it's reproducible.

Giny that is awesome

Mine worked too!!!!

So I tryed 5 of the proposed reagents, and what I saw was that some TX were better crosslinked with one agent and others with another.
Now I am trying to figure out a way to diagnose the xlink efficiency before I do the freaking 4 days of ChIP.
Do you know if it is possible to western blot xlink samples?


way to go Giny!

[giny]

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?

[Radish]

giny, on Feb 26 2010, 01:33 AM, 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.

[annabellak]

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%).

[giny]

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.

[KPDE]

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.

[Radish]

annabellak, on 08 March 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%).

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...

[kika]

so, Radish... what 5 regagents did you choose and why?, and is it possible to western the IPs??