anti-HA immunofluorescence on HEK 293 cells - (Aug/27/2018 )

Hello,

I've been trying to perform immunofluorescence on HEK293 cells overexpressing HA-tagged proteins.

I tried anti HA 12CA5 (Roche) 1:100, anti HA (C29F4) rabbit mAb from Cell Signalling (1:1000 and 1:500) and anti-HA (6E2) mouse mAb from Cell signalling (1:100).

I performed fixation with PFA 4% for 15 min at rt, then I blocked with 5% NGS, Triton 0.3% in PBS for 1 h at RT. The incubation with the primary antibodies was done either overnight at 4 degrees or overnight at rt, followed by  incubation with secondary antibodies (Alexa Fluor conjugated from Invitrogen) for 2 h at rt. I used Triton 0.3% also during the washing steps.

The antibody from Roche doesn't seem to work at all, while the others give a very faint signal.

I also checked the expression of my constructs by Western blot and they are definitely expressed.

Does anyone have experience with these antibodies or any tips to improve the staining?

Thank you!

How do your cells look after all the steps - 293 are notorious for lifting off during the washing steps, so you are likely losing a large proportion of the transfected cells.

Hi,

thank you for your reply.

I seed my cells onto polilysine-coated coverglasses and they adhere quite well.

I already performed immunofluorescence on transfected  HEK293 using other antibodies with no issues.. I'm encountering problems only with these anti-HA.

I note that for the Roche antibody you are using 1:100 dilution - this would give you 50 ug/ml. They recommend 1-10 ug/ml for IF. 

You may be over-fixing the cells. Try using ice-cold 2% PFA for 15 min. You can also try acetone or methanol, or acetone:methanol fixation (cool at -20, add to cells 20 min at -20, then wash off. If using acetone, you don't need the triton step).

Also check that the PFA has been prepared and stored properly.

Thank you for the advice. I will try to change the fixation step and see how it goes

So you used the same antibodies for Western blot and it was able to detect?