Hello everyone,
I am wondering if anyone knows if there is anything against using a mouse primary antibody on mouse tissue for ChIP. I know that using same-species antibodies for IF/IHC isn't ideal due to potential excess background, but is this the same case for ChIP analysis? The antibody is question is the cell signaling Myc-tag antibody and I am hoping to use mouse tissues in an upcoming ChIP experiment. Has anyone else run into any similar issues in the past?
Thank you,
Beatriz
You can refer to the links below to obtain important informations:-
1. http://www.abcam.com/protocols/mouse-on-mouse-staining-protocol
2. http://www.activemotif.com/catalog/494/bridging-antibody-for-mouse-igg
your targets are not an intact mouse tissues or cells, but a chromatin, and the bounded proteins on it. this is totally different from a IHC/ICC. i am sure u can use mouse monoclonals.
You can try as Dr.Lehotzky said. However, I am not so much confident because although its chromatin-bound protein yet the epitopes are same for all experiments. You should use the negative control the mouse IgG. I apprehend the difference will be smaller b/w IgG and antibody. But, if ultimately it works, plz let us know. That's a interesting and important observation irrespective of +ve or -ve.
I don't believe there is going to be any difference.
I shouldn't matter that the antibody is from mouse, it is just an antibody against MYC tag (wich is not an endogenous antigen in mouse). So your sample will only have MYC tag in your protein of interest and that will be the only thing recognized by it.
When performing ChIp you use protein G o A to pull down the antidody. Protein G or A binds a lot of different antibodies from a lot of different species so the background should be simmilar. It might be more important to have a very nice anti-MYC antibody (a monoclonal).
Finally depending on the ChIP protocoll you are using you might have only the chromatin and some minor contaminants from cell debris, It should have less background than an IP where you have a whole cell lysate.
Dear Beatriz,
I also believe that it should be feasible.
May be you may pre -clear the chromatin by incubating it with proteinG/A before the addition of the Primary Ab.
This should put you completely in a safe side I guess.
Good luck
Carmelo
Hello All,
Thank you so much for your input. After much scouring, I remembered a paper I read earlier this year, in which the authors used a mouse antibody on mouse tissue cells. I've linked the paper here. I will be trying this ChIP within the next 2 months or so, accounting for the upcoming holiday. So, I will be sure to post back with how that works out.
I really appreciate all your input on the topic,
Thanks all of you so much,
Beatriz
Paper: http://nar.oxfordjournals.org/content/early/2015/10/06/nar.gkv1023.full.pdf+html
Hi Beatriz,
If you want to look for or detect a protein in mouse tissues you can/should use primary antibody raised against mouse protein, it can be raised in any organism. if you are doing ChIP you can use mouse antibodies raised in mouse or any organism since for ChIP you use protein A to pull down rabbit raised antibodies, protein G to pull down goat/mouse raised antibodies, you can use a mixture of proteinA/B both too. it works fine. since IgG don't potentially binds to chromatin. so you don't expect background from there.
Goodluck!
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