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Thank you so much for your previous help. I have another inquiry now. I’m trying to isolate nuclei from mammalian cells and would like any advice on an antibody suggestion for this and more so for identifying said pull down by western. I have tried Histone Abs and would like something that is a bit bigger than 17 kDa but also easily distinguished from antibody chains that would be present in the pull down sample. And secondly, a good antibody that is polyclonal for identifying the presence of mitochondria in the above pull down sample, with same criteria as the Nuc western detection antibody described above.
Thank you so much.
Asked on Aug 07 2012
Thank you very much for contacting us with your questions.
Will you be trying to isolate intact nuclei from mammalian cells? I'm not sure if there is an antibody that would be good for this, butthecell fractionation kit ab109719 that I mentioned beforemight be suitable. I'll need to check with the lab whether the nucleiu will still be intact using this kit, if that is your end goal.
To check the efficiency of a nuclear fractionation, I would recommend probing for either lamin B1 (66 kDa) or TATA binding protein (38 kDa). Both of these proteins will run higher than the histones but should be far enough away from the IgG heavy and light chains around 25 and 50 kDa. For mitochondria, I'd recommend a VDCA1/porin antibody (31 kDa), though this may be too close to the 25 kDa light chain band. You could also probe for COXIV(16 kDa) but this protein may be smaller than what you'd like. What species will your samples be from? I can find specific antibodies that are known to react with this species.
I'm not sure if you would be interested inthis, but we do have secondary antibodies that only bind to native primary antibodies so that the denatured chains won't be visible at 25 and 50 kDa following an IP. For anti-mouse IgG, we have ab131368-
For anti-rabbit IgG, we have ab131366-
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.
Answered on Aug 07 2012