Hi All,
I have been attempting intracellular staining of a methyltransferase and have been running into issues with consistency. I have attempted sequential staining with a PE secondary and have also tried conjugating the antibody with a Zenon labeling kit.
My perm and fixation buffer are from biolegend.
Does anyone know if I could achieve better results with a FOXP3 perm buffer? Or is there another method I should be using? The protein should be in the cytoplasm but I can’t find any publications for staining this particular type of ptotein.
Also, has anyone ever used an isotype control for IC staining? I’ve always been advised not to but I have been told to do it for this stain and I don’t understand why (or why not for that matter).
thank you for your help!!!
If your methyltransferase is nuclear component then FoxP3 buffer will be helpful and if it's cytoplasmic then any perm/fix agent (methanol based) will be good.
I am not very supportive to use isotype now, all litterature suggest to use FMO rather than isotype. But in your case (if performing secondary staining) secondary antibody alone will work to nullify the non specific.
If you are using tagged primary antibody then you need to find an internal negative control for he same.
Attaching a good article (an old one) for methyltransferase flowcytometry. All the best.
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