I am doing ChIP qPCR using HeLa cells to see if my protein binds to a promoter region. As negative control primers I've been using primers from various companies such as Active Motif and SA Biosiciences. However I get signal when I do the qPCR using these primers. The signal is as strong or sometimes even stronger than my target of interest. I've done dilution series of my DNA and still have problems. The protein I am pulling down is not a traditional DNA binding protein like a Transcription factor. Is it possible to exclude these negative control primers or are they necessary? I've seen people publish without negative control primers.
Hi Dudi,
exactly because your protein is not a traditional DNA biding protein, you definitely need a negative control primers to show differential and specific pattern of biding. If you wish you can try to use the primers we used in our "scanning" ChIP-PCR (https://www.researchgate.net/publication/234061220_Direct_Transcriptional_Regulation_of_Human_Hepatic_Cytochrome_P450_3A4_%28CYP3A4%29_by_Peroxisome_Proliferator-Activated_Receptor_Alpha_%28PPAR%29?ev=prf_pub), targeting untranslated DNA regions (untr5).
But, perhaps, the phenomenon which you observe has indeed a different explanation - your factor binds just any DNA without being specific for some sequence?
Article Direct Transcriptional Regulation of Human Hepatic Cytochrom...
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