Anti-RNA polymerase II CTD repeat YSPTSPS antibody - ChIP Grade (ab26721)

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.

Dear Abcam Abreviews Team

I attached the JPG file in this mail.


I am sorry bothering you again.

ANSWER:

Thank you for sending your western blot image again. I have included it in your Abreview which is now published on our website.

Please let me know if you need anything further.

former lots worked well in ChIP this lot GR 33496-1 gives not good results samples: human prostate RT-PCR enrichment used to be 4 CT previously now no enrichment seen (with or withour treatment), done 2-3 times no change in protocol or samples Ab: 1 ug per reaction    

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxx. The estimated delivery date is xxxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.