Has anyone ever tried to stain 2 different proteins in a single sample, while they both originate from he same species (1 of them conjugated though)?
I have 1 primary antibody (host: mouse) which I intend to bind to a secondary anti-mouse antibody, and I have 1 conjugated antibody (host: mouse) and I need to stain them both in the same sample. Of course I make sure the 2 fluorophores are different, but should I be worried that the sec. antibody might bind the the conjugated complex??
thanks
Yes, your secondary antibody is going to bind to both primary antibodies. You should change your incubation order: incubate your sample with 1. unconjugated primary Ab against protein A, 2. secondary detection Ab, 3. directly conjugated primary ab against protein B. That might work.
Thank you very much - and I should wash the cells between the sec-AB incubation and the conjugated-AB incubation right?
Yes, of course the anti-mouse can bind any mouse antibody in the assay. Even in the order suggested by Kirstin, there is a risk that the anti-mouse already bound can capture the conjugated mouse antibody (if one of its binding sites is still free). Perhaps thats why she says this variant might work, but this is not 100% sure. I would suggest to search for two conjugaed mouse antibodies or an antibody from a different species and a secondary reagentewithout species cross-reactivity to obtain a cleaner experiment.
yes I understand her suggestion is not a guarantee and surely the best way to be avoid such risk is to purchase different antibodies..
thank you
That is exactly what I meant, thank you Gertrudis! The other possibility is to directly conjugate you primary Ab, there are easy to use fluorescent labeling kits out there.
I think the only way to know is to try staining the samples with 2 primary antibodies separately, then incubate them both with secondary antibodies (1:1000-1:2000, 0.5-1 ug/ml) at room temperature for an hour. You would easily tell if the secondary antibody binds to the conjugated primary. There is a change that the secondary antibody only bind to un-conjugated primary antibody, because the fluorophore conjugation may interfere with the binding from the secondary antibodies.