I would like to know if there is a simple approach to show whether or not a primary polyclonal antibody is not specific enough for an immunofluorescence experiment? I am using a anti-PECAM1 polyclonal antibody with HUVECs. In a separate culture, I am culturing a cancer cell line. I expect the HUVECs to stain positive for PECAM1 but the cancer cell line is also coming up positive for PECAM1, which is an unexpected result, and one which I highly doubt. Is there an approach for showing either that the cells are indeed producing PECAM1 or that the primary antibody is not specific enough? My other concern is perhaps something in my protocol is artificially creating sites for my primary antibody to bind. If need be, I will consider purchasing a monoclonal antibody. Could anyone recommend any monoclonal PECAM1 antibodies (company or clone)?
The experiments I am doing are in cell culture well-plates. Here is my protocol in brief:
1. Fix in 4% paraformaldehyde for 1 hour.
2. Wash with 1X PBS
3. Permeabilize with 0.2% triton X-100 for 15 minutes.
4. Wash with 1X PBS
4. Block with 5% serum
5. Incubate with 2 ug/mL primary antibody in 1X PBS with 5% serum.
6. Wash with 1X PBS
7. 5. Incubate with 10 ug/mL secondary antibody in 1X PBS with 5% serum.
8. Wash with 1X PBS
9. Stain with DAPI.
10. Wash with 1X PBS.
I often leave the cells in PBS to visualize under the microscope, or mount and coverslip if I want to view much later.
Any assistance is greatly appreciated. Thank you.
Hi Sebastian,
Your approach of using two types of cells is good (the ideal is testing the antibodies in cells not producing the target protein like knockout models). Signal came the same in intensity for both cell types? If it is like that I would check a bit in the IF protocol.
I just search for this Pcam1 protein and states it is a precuror for cell adhesion molecule which I guess will be enriched in subcortical domains when attached to permisive substrates. In this case I would recommend to reduce either the time or the detergent concentration for permeabilization step. I know these permeabilization parameters are very variabnle and values come from preset protocols in any lab but in general 0.1% Triton for 5 minutes should be enough for all mammalian cells.
Also be careful with the use of serum as blocking agent.It is a common "contaminant" for some antibodies. Try blocking with BSA 3-5% in PBS 1X and mix the antibody with a 1/10 dilution of this Blocking agent (BSA 0.3-0.5%). If something in the serum is depositing on top of your cells and this interacts with your primary antibody would explain why you get signal on both types of cells.
Hope these tip may help.
Good luck!
Me again,
I don't think PFA autofluorescence may the problem here but since the fixation you use is quitenlong you may also think in a brief quenching step with NH4Cl or Glycine to kill possible paraformaldehyde autofluorescence.
Hi,
What type of slide do you use,
sometimes the slide will give you autofluorescence.
I use primary and secondary Ab in just PBS.
greetings,
Johan
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