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Trouble validating in multiple cell types

H3 H3K27me3 H3K4me3 RPL30 GapDH

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#1 nleboarder

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Posted 25 March 2013 - 03:15 PM

I have a frustrating problem. I am using 2 cell types, fibroblasts and a B cell tumor cell line. I am doing ChIP for H3, H3K27me3 and H3K4me3. The validation works perfectly in all of my fibroblast samples, but completely fails in all of my B cell samples! For validiation for H3 I am using RPL30, for H3K27me3, MyoD1, and for H3K4me3, Hoxc11. Why does it work great for fibroblasts and not B cells?

Also, I have read that GapDH is considered a positive control for H3K4me3. I used it as my negative for enrichment and it works really nicely. In fact, when I am using my fibroblast cells, if I compare enrichment between GapDH and Hoxc11, I get on average around a 200 fold difference! (but essentially NO difference in B cells....errrrr)

Anyone out there have an issue validating in multiple cell lines?

#2 jerryshelly1

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Posted 27 March 2013 - 07:30 AM

Are the cell types you are using from the same species? Maybe your primers are for human and your b cells are mouse. What about of your Antibodies.





Also tagged with one or more of these keywords: H3, H3K27me3, H3K4me3, RPL30, GapDH

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