Anti-RNA polymerase II CTD repeat YSPTSPS antibody [4H8] - ChIP Grade (ab5408)

I found out ab5408 has two different size on the website. Please check the attached file and let me know if you have two kinds of products or it's an error.

Best regards

ANSWER:

Thank you for your message.

I apologize for any confusion. I have reviewed the online datasheet and can understand your question.

In this case, I can confirm you would be getting equal amount of antibody from both selling sizes as one size is in volume and the other size is in mass. This is due do this due to change in concentration. Both are the same product.

I have forwarded your feedback to my colleagues and the datasheet will be updated shortly to avoid any further confusion.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

I really appreciate your help and would like to receive the replacement. We will test these three antibodies along with some other antibodies for ChIP, and I will keep you posted about the result.

Once again, thank you very much for your help!

Have a great day-

ANSWER:

Thank you for your reply.

I'm sending the free of charge vials of ab5408 and ab5131 on the order ***, which should arrive tomorrow. The lot of ab5131 that I had intended to send is unfortunately no longer available, so I'm sending the lot ***, which is the only other lot that we have in stock but I am hopeful that it will work better. I hope that this will be acceptable.

The vial of ab5095 is also coming free of charge on the order ***. This is transferring from our UK office and should arrive early next week.

I look forward to hearing from you and I appreciate you keeping me posted! Let me know if you need anything else. Have a good day, too!

Thank you very much for your help. Could you please send me the attachment again?

Thanks!

ANSWER:

I am sorry I forgot to attach the data in my previous email. It is now attached.

Sorry about that!

Thanks for your email and infor regarding freezing antibodies.

Here are basic infor about our ChIP procedures:

1. what dilution or concentration you're using for each antibody in ChIP?
We used 2 ug and 4 ug for 5 million cells in 1ml of ChIP binding buffer.

2. What kind of cells are you studying, and is the chromatin fresh?
U2OS cells, freshly corsslinked, used H3K36me3 as a positive control.

3. What genes are used as the positive controls?
We tested both total Pol II, S5P and S2P. We used Myc gene promoter 100bp, gene body 3K and 6K downstream of TSS as positive controls for them. At all the regions we only observed IgG levels of signals (0.0002-0.001% of Input). I was expecting 0.2-1% of input signals, based on our experience with the same Abs of previous lots.

ANSWER:

Thank you for sending the additional information, and I apologize for the extended delay getting back to you. Our ChIPscientist has been out of the office, but I have received further information from him regarding these antibodies.
I have found lots for each of these antibodies that will be suitable for your studies.


Please let me know if you approve of these, and I will send them out promptly. The vials of ab5131 and ab5408 will be sent overnight and delivered tomorrow, but the vial of ab5095 will take a few extra days to transfer from our UK office.

I apologize again for the delay, but I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that we can do for you.

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.