Human GAD1 ChIP Primer and Probe Set (ab85780)

I attended your ChIP webinar last February.I am currently optimizing a ChIP-seq protocol with my cells (murine primary cells), with an antibody against Pol-II (product ab817). Before going on with the sequencing part, I would like to make sure that my immunoprecipitation worked well. As you suggested in your Webinar, I am looking for positive and negative control loci that I will test first by qPCR. Would you have any suggestions for negative control regions for Pol-II binding ? Have you ever used telomeric or intergenic regions for example, or would you rather recommend non-expressed genes in the tissue under study ? I also saw in some cases people using negative ascites as control. What is that for ?

Thank you very much in advance for your help.

ANSWER:

Thank you for contacting us.

We have tested some of our RNA polymerase II antibodies against both inactive genes and the 3' end of active genes. Below is a link to the antibodies.

http://www.abcam.com/RNA-polymerase-II-CTD-repeat-YSPTSPS-antibody-ChIP-Grade-ab26721.html
http://www.abcam.com/RNA-polymerase-II-CTD-repeat-YSPTSPS-antibody-4H8-ChIP-Grade-ab5408.html

We also stock a number of pre designed primers to human regions in our catalogue.

http://www.abcam.com/index.html?pageconfig=resource&rid=10600

Reviewing the antibody datasheets there are a number of regions and primer sets that might be suitable.

MyoD1
http://www.abcam.com/Human-MyoD1-exon-1-ChIP-Primer-and-Probe-Set-ab85776.html

GAD1
http://www.abcam.com/Human-GAD1-ChIP-Primer-and-Probe-Set-ab85780.html

AFM
http://www.abcam.com/Human-AFM-exon-1-ChIP-Primer-and-Probe-Set-ab85778.html

Factor VIII
http://www.abcam.com/Human-Factor-VIII-exon-1-ChIP-Primer-and-Probe-Set-ab85775.html

I have not seen people using negative ascites as a control but I assume this is a negative control for the IP part of ChIP. It is also possible to use no antibodies or a non-specific antibody that is from the same host species and isotype. e.g. use a rabbit IgG if this matches your primary antibody.

I hope this helps. Please let me know if there is any other information you need.