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Customer inquiry regarding anti-histone H3 antibodies. Study is investigating methylation status of Histone H3, specifically on Lysine 9 (K9). Products I am already considering are ab70550 (anti-histone H3), ab1220 (anti-histone H3 di methyl K9), ab8898 (anti-histone H3 tri methyl K9), ab3790 (anti-KMT8/Riz1/Riz2). What protocol do you recommend in regards to preparing a cell lysate for detecting histone H3 via western blot? What is the minimum amount of cells that should be used? Why would one investigate these antibodies together in one study?
Asked on Nov 21 2012
Thank you for contacting Abcam.
We recently spoke on the phone regarding a Histone H3 methylation study. Below I have summarized our conversation and answers to your questions.
For detecting nuclear proteins via western blot we recommend using a RIPA buffer or cell fractionation protocol in preparation of your cell lysate. I attached our protocol book to this email for information regarding specific cell lysate preparation procedures. Below are the fractionation kits that we talked about during your phone call:
Nuclear Extraction Kit (ab113474) https://www.abcam.com/episeeker-nuclear-extraction-kit-ab113474.html
Histone Extraction Kit (ab113476)
The Histone Extraction Kit can be used to extract histones from as low as 1 million cells or 1 mg of tissue as stated on the datasheet. These lower limits are good guidelines for the Nuclear Extraction Kit as well.
Since ab1220 (anti-histone H3 di methyl K9) is the only antibody in your provided list that is raised in mouse (the others are raised in rabbit), we discussed substituting the rabbit monoclonal antibody ab32521 (https://www.abcam.com/histone-h3-di-methyl-k9-antibody-y49-ab32521.html) (anti-histone H3 di methyl K9) so you can use the same secondary for each antibody. Since ab32521 is a rabbit monoclonal antibody, it also qualifies for our RabMAB promotion. Details of this promotion are at the end of this email.
A list of our anti-rabbit secondary antibodies is given through the link below, depending on the detection method and conjugation that you prefer:
Together these antibodies could be used to detect the presence of histone H3 in your samples as well as the methylation status of Lysine 9. To complete the spectrum, Abcam also sells an anti-histone H3 mono methyl K9 antibody (ab9045, https://www.abcam.com/histone-h3-mono-methyl-k9-antibody-chip-grade-ab9045.html). As we discussed, ab3790 (anti-KMT8/Riz1/Riz2) detects the presence of PR domain zinc finger protein 2, which is a histone methyltransferase that specifically methylates Lysine 9 of histone H3 according to SwissProt (http://www.uniprot.org/uniprot/Q13029), thus explaining its connection to the anti-histone H3 antibodies that detect the methylation status of Lysine 9
Answered on Nov 21 2012