ChIP Normalization - (Mar/25/2006 )

Hi All,

I have question regarding normalization for ChIP. I'm doing ChIP for 7 different cell lines with different growth rate. Some of the protocols I've seen recommended getting a count on cell number for all the cell lines before cell lysis for normalization. However, I've noticed that after crosslinking, it's difficult to get a correct cell count using the Coulter counter since cells seem sticky and clumpy. If anyone has done this for a lot of cell lines at one time, please tell me what is the best way to count cell after crosslinking.

I've also seen protocols where people do protein assay and start out with same amount of protein lysates for IP for all cell lines. For me, this is a much simpler way especially since I've 7 different cell lines to work on. However, I don't know if this is the standard way of normalization or not.

Any input on this is greatly appreciated.

Thank you

In the past I trypsinise cells into suspension count and then crosslink in suspension. Which would have allieviated the problem of clumping. Although you could say the the process of trypsinisation could affect the chromatin in some manner. I think protein normalisation is becoming a standard protocol to perform.

Nick