Immunofluorescence: simultaneous polyclonal and monoclonal staining to same prot - (May/03/2013 )
I am looking at the localization of Protein X during UV irradiation.
I noticed that increased fluorescence of protein X using a monoclonal antibody within 30 minutes of UV irradiation. Since the increase is in a very short time period I do not think it is due to increased expression or decreased degredation (if so, that itself would be a lone paper).
I am hypothesizing that since I used a monoclonal antibody, UV irradiation caused a shift in protein folding or uncovering of the antigen binding site, hence the increase in immunofluorescence signal.
So I am planing to stain with a polyclonal antibody.
I was wondering, if I simultaneously incubate the samples with mouse polyclonal and rabbit monoclonal to the same protein, would I be too optimistic in thinking I would see good staining? Should I just do the two antibodies separately?
And if I go ahead with simultaneous staining, should incubate the two primary antibodies at the same time or one after the other? (monoclonol first or polyclonol first?)
I tried to search the literature for any suggestions but I could not find any attempts such as this.
Should work fine, just watch out for compatibility of the secondaries.