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Normalization problem


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#1 Jeannine

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Posted 01 November 2010 - 03:34 PM

Hi all,

I'm doing ChIP assay since a few month and have successfully optimized the protocol in my cell lines. I'm using FLAG beads (M2 resin) and cells stably expressing a FLAG-tagged transcription factor. To control the ChIP, I'm using primers flanking a known binding site and normally get a 15-18 fold enrichment and nearly no background. I recently made new stable cell lines, with pointmutations in the transcription factor. To test if the mutations have an effect on DNA binding I want to do a ChIP assay with these cells and compared them to the wt. I tried to pick clones, that have the same expression of my protein, but unfortunately the amount of my transcription factor still differs in the cell lines.
Can I still use these cells for ChIP assay, and if yes can I normalize the enrichment dependent on the protein conc.?
As a example, these are the results of my first ChIP with the new cell lines (note: the enrichment is normalized to input and background):
Enrichment wt: 18-fold
Enrichtment mutant: 17.8-fold
Expression wt: 1-fold
Expression mutant: 1.5-fold

Is the "real" enrichment of the mutant 17.8/1.5 = 11.8 or is this calculation wrong?

Any suggestions what to do?

Thanks,
Jeannine

#2 KPDE

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Posted 02 November 2010 - 08:07 AM

Hi all,

I'm doing ChIP assay since a few month and have successfully optimized the protocol in my cell lines. I'm using FLAG beads (M2 resin) and cells stably expressing a FLAG-tagged transcription factor. To control the ChIP, I'm using primers flanking a known binding site and normally get a 15-18 fold enrichment and nearly no background. I recently made new stable cell lines, with pointmutations in the transcription factor. To test if the mutations have an effect on DNA binding I want to do a ChIP assay with these cells and compared them to the wt. I tried to pick clones, that have the same expression of my protein, but unfortunately the amount of my transcription factor still differs in the cell lines.
Can I still use these cells for ChIP assay, and if yes can I normalize the enrichment dependent on the protein conc.?
As a example, these are the results of my first ChIP with the new cell lines (note: the enrichment is normalized to input and background):
Enrichment wt: 18-fold
Enrichtment mutant: 17.8-fold
Expression wt: 1-fold
Expression mutant: 1.5-fold

Is the "real" enrichment of the mutant 17.8/1.5 = 11.8 or is this calculation wrong?

Any suggestions what to do?

Thanks,
Jeannine


I don't think you can quantitatively normalize by the protein expression (maybe qualitatively). There is likely error in that the two measurements (ChIP and protein expression) have two different dynamic ranges.

You might be able to get quantitative results using EMSAs, though I don't have experience with them and don't know how noisy they are.

Edited by KPDE, 02 November 2010 - 08:15 AM.





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