Anti-KMT1C / G9a antibody - ChIP Grade (ab40542)

I would like to know what the PCR primer sequences are that you use to check the ChIP-ability of each batch of G9a antibody, in order to use as a positive control.

My samples will be from human and mouse.

ANSWER:

Thank you for your enquiry. I apologise for the delay in responding to your message. It is taking some time to obtain details from the originators of these antibodies.

I do now have the ChIP information for G9a antibody ab40542. ChIP for G9a antibody was performed through two different approaches. First, Gal-G9a fusion was expressed in Xenopus oocytes. ChIP was then carried out using a reporter with four Gal4 binding sites as described in a reference paper:

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2005, p. 2525–2538 Vol. 25, No. 7 Relationship between Histone H3 Lysine 9 Methylation, Transcription Repression, and Heterochromatin Protein 1 Recruitment M. David Stewart, Jiwen Li, and Jiemin Wong*

Second, the laboratory found that G9a was recruited to target genes repressed by unliganded thyroid hormone receptor (TR) in HeLa a2 cells, which constitutively expressed a Flag-tagged TRa.The primer sequences for ChIP analysis of TR target gene (deiodenase one) are 5'-GGAGGCCAAGGCGGGTAGGTCATCT-3¡¯ and 5¡¯-CCGGGTCAGGGGAAGGAGTCAGGTCA-3¡¯. However, since in this case G9a was believed to be recruited to the deiodinase one gene by unliganded TR (unpublished data). Unfortunately, they are not able to confirm if this set of primers can be used as positive control for G9a ChIP.

I hope this information is helpful. If you have any further questions, please do not hesitate to contact me again. As soon as I recieve further information on the SUV39H1 antibody (ab12405), I will send this information on to you.

what is the chance that this G9a antibody will recognize mouse G9a? I would like to try this Ab for ChIP analysis. Please reply asap.

ANSWER:

Thank you for your enquiry.

This antibody was raised against a recombinant fusion protein, corresponding to amino acids 831-1001 of Human G9a. I have performed an alignment of this region with the corresponding mouse homolog. This has a very high degree of sequence similarity. Please see alignment below;

http://www.ebi.ac.uk/cgi-bin/jobresults/clustalw/clustalw-20061020-10153699.aln?

I would consider this very likely to react with the mouse homolog.

It has been shown to work by chromatin IP. Should you purchase this antibody we would very much like to hear how it performed by way of our Abreviews system. By providing feedback and leaving an Abreview you can earn Abpoints that can be redeemed against various items such discounts against future purchases, Amazon.com vouchers etc.