I'm getting a lot of non-specific staining with a polyclonal ERa antibody I'm using. There's so much of it that I'm beginning to think that my incubation period (48 hrs at 4C) is too much. Previously, I tried a monoclonal ERa antibody that I only tried with a 24 hour incubation period before I ran out of it. I got light cell staining so I decided to try a different antibody (polyclonal) and increase the incubation time to 48 hours. It worked, but my ERa stain looks like dapi. Any input would be appreciated.
Hi Marcia
48 hour incubation is rather long. I assume you purchased these antibodies from a commercial company. In which case, the company should have supplied you with the information on optimal reaction conditions (temperature, incubation time, pH, buffer etc) to get the 'best' results. If they didn't, you should contact them and ask them. If that doesn't work, I suggest you set up an experiment and collect aliquots of reaction mix at intervals of 12, 24, 36 and 48 hours, stain and examine them. You should get an idea which incubation time gives you the best results.
Good luck
Siva
Hello Marcia Chavez
Polyclonal antibodies are generated by injecting an animal (most commonly a rabbit or goat) with the antigenic preparation of interest and harvesting the animal's serum once an immune response is detected. The serum is subjected to antibody purification and sometimes to differential adsorptions to eliminate unwanted reactivity, but it always comprises a spectrum of antibody molecules originating from multiple unrelated antibody-producing cells. Therefore, termed polyclonal. The specificity of a polyclonal antibody preparation is highly dependent on the purity of the initial antigenic preparation and how extensively adsorbed it is. Obtaining highly specific preparations is difficult, and background problems can be troublesome. Further, because the antibody response is variable over time and from one individual animal to another, complete standardization of antibody composition is not possible.
Usually, the primary antibody incubation time is 1 to 2 hours at room temperature or overnight at 4 °C in the dark. If you are using monoclonal antibody, you can improve the cell staining by adjusting the antibody dilution. Also, it is always possible to enhance the intensity of the signal as long as the background remains low. So, you should try blocking for 1 hour at room temperature. You could use (1%–5%) Bovine Serum Albumin (BSA) or serum as blocking buffer. Use blocking serum from a different organism than the primary antibody.
So, I suggest you use monoclonal antibody. You can improve your staining by adjusting the antibody dilution and keeping the background signal as low as possible by using the blocking buffer mentioned above.
Best.
Hi,
assuming your target is well-expressed in the used cell line, you shouldn't have problems with antibodies registered for IF. Personally, I incubate cover-slips for 2h at room temp or overnight at +4°C. Dilute the antibody in 3% BSA (protease-free) in 1:50, 1:100, and 1:200 and test for the optimal antibody dilution. You should also include negative staining control (entire procedure without primary antibody) for possible unspecific binding of secondary Ab.
For: "It worked, but my ERa stain looks like dapi." I assume it stained only the nucleus, and that is not what you expected.(?). I had a problem like this, and the problem was incomplete nuclear permeabilization. As soon as that problem was solved IF worked perfectly.
In regards to mono- or poly-clonal Ab, polyclonal is more advantageous for IF as they can recognize multiple protein conformations (in IF proteins are not denatured like in WB) but I never had a different result using both for IF.
I hope I managed to help you with your problem,
Good luck