Negative control primer for ChIP - (Apr/10/2007 )
Hi everybody,
I have recently started working on ChIP with NF-kB. Usually, I treat cells with TNF or LPS and then look at NF-kB binding to promoter regions. I get good enrichnment with NF-kB antibody versus normal mouse serum. However, the problem is that if I use GAPDH (Upstate) as a negative control gene - I detect NF-kB binding to it as well.
So, I am not sure if NF-kB really binds to GAPDH and I should choose another negative control gene like actin, or something else.
Please help with this and if you have nucleotide sequence for good negative control primers I will really appreciate it!!
Thanks in advance.
Activemotif's Chip-it kit uses the following negative control primers
Neg For: 5´-ATGGTTGCCACTGGGGATCT-3´
Neg Rev: 5´-TGCCAAAGCCTAGGGGAAGA-3´
According to the instruction: the negative control primers flank a region of genomic DNA between the GAPDH gene and the chromosome condensation-related SMC-associated protein (CNAP1) gene. The PCR product generated with these primers is 174 bp long. Because the genomic DNA flanked by these primers (cytogenetic location 12 p13.3) should not be bound be transcription factors, this locus should not be enriched by ChIP. Thus, PCR with the negative control primers should give rise to equivalent amounts of product whether it is performed on DNA isolated by immunoprecipitation with anti-RNA pol II or with the Negative Control IgG.
I have recently started working on ChIP with NF-kB. Usually, I treat cells with TNF or LPS and then look at NF-kB binding to promoter regions. I get good enrichnment with NF-kB antibody versus normal mouse serum. However, the problem is that if I use GAPDH (Upstate) as a negative control gene - I detect NF-kB binding to it as well.
So, I am not sure if NF-kB really binds to GAPDH and I should choose another negative control gene like actin, or something else.
Please help with this and if you have nucleotide sequence for good negative control primers I will really appreciate it!!
Thanks in advance.
Hi Fiji,
What means "I detect NF-kB binding to it as well" ? - In ChIP you usually have background, so if you just see some PCR signal that doesn´t mean
NF-kB is binding to GAPDH. If you see a stronger signal with normal mouse serum compared to NF-kB antibody this might also be okay, because background with mouse serum might be stronger.
You should compare the GAPDH enrichment to the enrichment of a positive target.
Thanks a lot for your answers!
What means "I detect NF-kB binding to it as well" ? - In ChIP you usually have background, so if you just see some PCR signal that doesn´t mean
NF-kB is binding to GAPDH. If you see a stronger signal with normal mouse serum compared to NF-kB antibody this might also be okay, because background with mouse serum might be stronger.
You should compare the GAPDH enrichment to the enrichment of a positive target.
GAPDH gene expression is known to be affected by TNF and LPS in some cases. So I was not very surprized to see how p50 and p65 (NF-kB subunits) bind to its promoter after stimulation (not as strong as with IL-8 promoter of course).
So that is why I kind of looking for set of primers that is in the region free of any transcriptional activty.