Hello,
I've been trying to perform immunofluorescence on HEK293 cells overexpressing HA-tagged proteins.
I confirmed the expression of my constructs by Western blot but the immunofluorescence does not seem to work (high background and non specific signal).
I tried anti-HA 12CA5 from Roche (1:100) which works well in Western blot but not in immunofluorescence, anti HA (C29F4) rabbit mAB (Cell Signalling) 1:1000 and 1:500, anti HA 6E2 mouse mAb( Cell Signalling) 1:100.
I performed fixation with PFA 4% for 15 min at RT and blocked with 5% NGS, Triton 0.3% in PBS for 1 hour at RT. The incubation with the primary antibodies was done either overnight at 4 degrees or overnight at room temperature. The incubation with the secondary antibodies (Alexafluor conjugated) was done for 1 h at rt. I also used Triton 0.3% during the washing steps.
Does anyone have experience with these antibodies or any tips to improve the staining?
Thank you!
I would say that your are using too much triton and too long. What you want is to punch holes in the membrane in order to allow the Ab to pass and reach its epitope. By incubating the cells with triton for several hours you literally destroy the membranes (plasmic and nuclear) and thereby alter the cell structure and as a consequence the quality of your staining.
After fixation, I would recommend you to perform a permeabilization in PBS 0.1% triton for 10 min at room temperature followed by 3 x wash 5' with PBS.
Block with PBS 2%BSA, 1H at RT. Rinse.
Primary Ab diluted in PBS 2%BSA, 1H at RT
wash 3x 5' PBS RT
2ndry Ab diluted in PBS 2%BSA,1H at RT
wash 3x 5' PBS RT
This should give you very good results.
Hi, if I were you first I would try to lower the incubation time with the primary AB. It will bind in 1h RT, or even in 20 min. I also tried 4°C O/N once, gave very high background. Also your permeabilization seems to be too harsh, 8-10 minutes 0.1% will do the job
Thank you Anand.
Someone else suggested me to modify the fixation step.
I will try and see how it goes
The anti-HA.11, clone 16B12, recognizes the HA epitope YPYDVPDYA. I have used many anti-HA antibodies including the 12CA5 clone you mention and none work so well as the 16B12 clone. If the epitope is inserted in a loop (not C-term or N-term tagging) I have not found any other antibody to work for immunofluorescence. For immunofluorescence, a 1:1000 dilution (1 microgram/mL final) as a good starting point (I get a modest perinuclear signal at 1:500 in control MDCK cells).
Formaldehyde can be used (2 to 4 percent) and permeabilization with either Triton X-100 or saponin work well.
If anyone has the hybridoma cells I would appreciate it, I was never able to find it.
Hi Dr Ryan Schreiner, do you think the CST HA antibody, C29F4 clone work well in WB?
Pinzhu Huang, the rabbit monoclonal C29F4 is new and I have not tested it yet. The current rabbit polyclonal I use is from MBL, http://ruo.mbl.co.jp/bio/dtl/dtlfiles/561-v4.pdf
The C29F4 looks promising also.
- Badges
- Science method