ChIP Chromatin Immunoprecipitation Lysis Buffers?

[dave2006]

Hi every1,
I have questions on cell lysis for ChIP.

After cross-linking my cells, what lysis buffer should I use?

In different protocols they use different lysis buffers. Some lyse their cells directly using 1% SDS-Lysis buffer but others lyse the cells and isolate the nuclei and afterwards lyse the nuclei.

Are there any reasons why?

Also which is better RIPA buffer or SDS-lysis buffer? Does it depend on the cell line? I am currently using transfected HEK 293 cells.

Thanks in advance

[rimapant]

Hi Dear,
Though I am new to this area but found very good concept about choosing the lysis buffer in abCam website. In short ,Ripa is more stringent than NP-40 or Triton-X100. It depends on your antigen. Read IP protocol in their website that will help you to make a decision.

[dave2006]

Thank you very much for your reply. i will read the protocol there.

thanks

[a05rahde]

hi, i work on preimplantation embryos. I would like to use ChIP for the analyasis of DNA methylation.. can anyone suggest how many embryos I need or how much DNA I need to analyse global DNA methylation?

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