I would like to study the total expression levels of a kinase that is located both in nucleus and cytoplasm. I don´t have many cells for the analysis that is why I would like to perform the detection using FC instead of WB. But I couldn´t find any conjugated antibody to detect this protein, therefore I am using a monoclonal antibody suitable for FC (mouse IgG1) and then a secondary antibody conjugated with Alexa Fluor 488 (goat anti-mouse IgG H+L) to detect the primary antibody bound. Could anybody tell me, which negative control / isotype control for this type of staining should I use?? Because I can detect my protein of interest, but I don´t know how much background I have...
I prefer to use unlabeled antibody (ex cloneY) to block binding of labeled antibody (FItc-cloneY) to determine background staining, You can block with an unlabeled irrelevant/different antibody to determine specificity of blocking. I add the blocking antibody in excess and don't wash it out when I add labeled antibody.
Hope this helps
Thank you Karen for your answer, but I am not sure whether I´ve understood you correctly. You mean, to use unlabeled antibody that doesn´t recognize the protein that I want to detect and then add the labeled antibody?So, in this case, would be to add mouse IgG1 that does not recognize anything, and then the secondary antibody labeled?
Usually we do an unstained control as well as a negative control stained with only the secondary antibody (AF488 in your case). Your secondary antibody should not bind to anything, so if you have staining, it's only non-specific staining (a.k.a background).
Hope this helps!
We follow a similar protocol in order to detect intranuclear proteins using unconjugated primary antibodies. Initially we were using secondary Ab only as Genevieve suggested, which is an acceptable approach to assess non-specific binding. Now we are using an isotype control antibody as you have suggested, which I believe is a better negative control. In your case, this would mean first staining with an isotype control mouse IgG1, followed by your usual Alexa Fluor 488-conjugated secondary antibody (goat anti-mouse IgG H+L).
Hello Ceri, thank you for your suggestion! I was doing that, but my isotype control, which was pure mouse IgG1, was giving me also a positive staining and I think it was a false positive. The thing is I am not sure if the unconjugated mouse IgG1 that I was using was a suitable isotype control for an intracelullar staining, that is why I was searching for alternative negative controls....
Ideally you should use an isotype control which is formulated in the same way as your test antibody. If your antibody is suitable for flow cytometry then I would expect the manufacturer probably also has a suitable isotype control antibody available. Another important consideration is concentration; check that you are using the same amount of isotype antibody per stain as you use of the test antibody. If possible, test out your antibody with a positive control (e.g. transfected cells) or negative control (e.g. following knockdown or comparing with cells known to lack expression) to verify the specificity of the stain. Good luck!
Thank you again Ceri. I will check that, otherwise if the isotype that I have is not suitable, I think I will just use the secondary antibody only....
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