EGFP protein location problem fixing with PFA - (Nov/24/2010 )
Hi,
Recently, when I do IF experiments, I find GFP tag without any target proteins always locates at centrosome though this kind of dot location is not so obvious, we still can see it. I fix the cells with cold-PFA on the ice for 10min then wash it with PBS 3 times for next step.
I did the experiments to fix GFP tagged target protein with cold methonal on the ice, but the signal is delocation. It should be a protein that locates on the cenrtrosme. However when I fix the cells with methonal I cannot see any signal in the centrosome. So now I don't know what I can do.
Does any one has any suggestion? Thank you!
both PFA and methanol should work fine. but I have never fixed with pure methanol. it was always 50-50 methanol-aceton. it worked even better than PFA
Curtis on Wed Nov 24 14:26:21 2010 said:
both PFA and methanol should work fine. but I have never fixed with pure methanol. it was always 50-50 methanol-aceton. it worked even better than PFA
Yes,it should be. But the fact seems not so satisfactory. 50-50 methanol-aceton cannot make GFP tagged target protein leave centrosme? I never tried this fixion.
Christina,
At present, PFA causes artefacts in some proteins' localization by immunofluorescence. Changing fixation to pure methanol solves the problem.
Best,
Jonas