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ChIP for a transcriptional coactivator


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#1 yds2

yds2

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Posted 19 April 2013 - 06:16 AM

Hi everyone,

I'm doing ChIP for a transcriptional coactivator. I overexpressed it in 293T cells and now I'm trying to optimize the protocol before I move onto in vivo experiment with embryonic mouse brain.

I fix the cells with 1% formaldehyde. After lysing the cells in a hypotonic buffer, I collect the nuclei, which then I lyse in a buffer with 1% SDS in it. After this step, I sonicate my samples, and dilute them 10X before IP.

My questions are:
1. Do you think 1% formaldehyde would be enough to cross-link DNA to the transcription factor and transcriptional coactivator? I'm just worried during sonication, the protein-protein-DNA complex will come apart.
2. Would 1% SDS destroy the cross linking between the two proteins in this complex? I tried to replace 1% SDS with 1% NP40, but sonication did not work.

I'm just worried that I won't be able to keep the whole complex cross-linked together during the protocol, especially at the nuclei lysis and sonication steps.

I appreciate all your suggestions!

Best,

#2 Vassil

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Posted 30 April 2013 - 05:27 PM

Hi,

You need SDS in order to shear your DNA. NP-40 does not do the job. 1% SDS is usually well tolerated in terms of epitope concervation. You should be careful not to over-sonicate, though. This could destroy your epitope and heat the proteins inducing degradation (always perform in ice-cold water). Also, make sure that there are no bubbles when you sonicate, as this could also destroy your epitope due to surface tension.

1. It is possible that 1% formaldehyde may not be able to attache the co-activator to the desired site. To address this problem, you can use protein-protein crossllinkers, which have longer arms, and then perform the 1% FA crosslinking to link the complex to the DNA. Alternatively, I use 4% Paraformaldehyde for 25min at RT and it works fine for me (do NOT exceed 30min).

2. You should use 1% SDS. Do not replace this component of your buffer.


Good luck!

Vassil




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