Hi folks,
I am recently working with Arabidopsis to look at the protein which has been reported to be at cell plate involved in cytokinesis with YFP tag. However, when I fixed seedlings which functionally expressed this protein fused with myc or YFP tag with 4% paraformaldehyde and used anti-myc antibody (9E10, sigma or C3956, Sigma) or antiYFP antibody (A1112, Invitrogen) to detect signal, I found the proteinwas no longer at cell plate any more, instead, it was only cytosolic (this protein has also shown to be in cytosol). Does anybody have experience with this case? Is there any paper reporting paraformaldehyde can affect localization of proteins? How can I do with immunostaining?
Thank you in advance!
Hi Feng,
I am not as proficient with plants, but there is a paper published in PLOSONE that does touch upon the topic. I've provided the link below:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135033
Have you tried other fixation methods? Can you use OCT for preserving the seedlings? Using frozen sections, the proteins are in their native forms, and that might give you the localization you are looking for.
Good luck!
Methods for fixing can affect localization of some proteins (or at least the appearance of said), and can effect efficacy of some stains. Trying different staining is a good idea. Similarly, antibodies can have different epitopes they recognize - if your antibody recognizes part of the protein that's buried at its binding interface, then you won't see it there.
You could also try to isolate the region you're interested in (e.g. the cell plate) through cell lysis, centrifugation, filtering, etc., then run just that part of the cell by western blot, and see if you detect the protein that way. If you can purify cell plates, and detect the protein there, then chances are it's a problem with your IF protocol.
Another thing to consider: the report you're referencing with different localization could, simply, be wrong. I'd try duplicating their procedure as exactly as possible - if you don't see it after a few tries, it could be that THEY were the ones having problems with their immunofluorescence protocol.
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