Hi - I am having issues with losing protein localization from native sites upon fixation and antibody staining. Was wondering if anyone has implementation protocol when they can precipitate the protein in situ and fix for subsequent immunostaining?
Hi Mukhtar,
TCA 2% is a powerful fixative. It preserves structures well but is not suited for all antigens. It seems to be the first choice if phosphorylation contributes to antigenicity. You should include the specimen type/whole mount, cell culture embedded and at least the antigen you want to stain. For getting more specific answers.
Cheers
David
Saw I posted this question a while back. David, I did try and unfortunately wasn’t suitable for my antigen; exocyst subunits. Anyway, resorted to tagging endogenous proteins using CRISPR/Cas9 and was able to study the dynamics of my complex in live cells instead. Cheers.
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