positive controls for CHIP - (Jul/03/2006 )
Hi,
I has been working on CHIP assay for a while. Unfortunately, I still got non-Ab background which has the similar brightness as the one with Ab added. I tried adding more salmon sperm DNA to block the beads. Also, I tried adding more chromatin for IP to get more enrichment of my +Ab sample. I also tried different PCR cycles...However, none of them works... The PCR products(+Ab and -Ab) alway appear together or disappear together.
I began to doubt about if the CHIP can work under my hand. Can anybody recommend some positive controls that definitely work for CHIP?
a lot of thanks,
-rena-
QUOTE (rena @ Jul 3 2006, 01:58 PM)
Hi,
I has been working on CHIP assay for a while. Unfortunately, I still got non-Ab background which has the similar brightness as the one with Ab added. I tried adding more salmon sperm DNA to block the beads. Also, I tried adding more chromatin for IP to get more enrichment of my +Ab sample. I also tried different PCR cycles...However, none of them works... The PCR products(+Ab and -Ab) alway appear together or disappear together.
I began to doubt about if the CHIP can work under my hand. Can anybody recommend some positive controls that definitely work for CHIP?
a lot of thanks,
I has been working on CHIP assay for a while. Unfortunately, I still got non-Ab background which has the similar brightness as the one with Ab added. I tried adding more salmon sperm DNA to block the beads. Also, I tried adding more chromatin for IP to get more enrichment of my +Ab sample. I also tried different PCR cycles...However, none of them works... The PCR products(+Ab and -Ab) alway appear together or disappear together.
I began to doubt about if the CHIP can work under my hand. Can anybody recommend some positive controls that definitely work for CHIP?
a lot of thanks,
Hi,
We have used an antibody to RNA pol II (Santa Cruz #sc-899) and/or to histone H3 (Abcam #1791) for positive controls in our lab. Both antibodies work very well in ChIP and IP a lot of chromatin (since both proteins are bound to the chromatin in high abundance). In addition, if you use primers for a gene which is inducible then you can see an increase in RNA pol II and sometimes a decrease in H3 upon induction of the gene. This way you can tell if your enrichment is real.
Joel
-KPDE-
Thank you very much, Joel. I do find anti-histone H3 (Santa Cruz) in my lab. I am going to try the CHIP again using the anti-histone H3 as a positive control.
-rena-