Problem isn't so much sensitivity/quantification (which is indeed trickier than for WB) as it is selectivity. Antibodies will likely interact with different epitopes in either method (i.e. different cross-reactivities vs native/fixed (IF) and denatured (SDS-PAGE/Western). And contrary to Westerns, IF does not allow to discriminate based on size, so that any signal will be a tally of signals that co-localize spatially. You might want to include siRNA controls or equivalent to validate your signals. Good luck!

Do you use a loading control for your western blots?

Yes, it is Vinculin for PXR and CAR nuclear receptor proteins.

Are you using the same antibody for western and IF?

Yes, is the same antibody /

In that case i can think of two things:

1. your secondary IF antibody detects something unspecific, you may want to try difrent secondary or secondary only and see if that solves the problem

2. In my opinion it is more difficult to get quantitative data from IF experiments, you have to make sure that you exposure times and settings are identical between your samples, i think i would trust western data over IF

Thanks Sylwia! Yes, I agree that IF is far less sensitive than western.

Problem isn't so much sensitivity/quantification (which is indeed trickier than for WB) as it is selectivity. Antibodies will likely interact with different epitopes in either method (i.e. different cross-reactivities vs native/fixed (IF) and denatured (SDS-PAGE/Western). And contrary to Westerns, IF does not allow to discriminate based on size, so that any signal will be a tally of signals that co-localize spatially. You might want to include siRNA controls or equivalent to validate your signals. Good luck!

Overall IF is a qualitative experiment (though you can quantity the IF intensity), and WB is more quantitative experiment. You can try antigen retrieval, that will help to open some epitopes of antigen for better binding of the primary antibody.

Dear Venc Cts ,

It is rather normal to have discrepant results using distinct techniques: you are quantifying different variables. Although it would be nice to obtain a similar result using both WB or IF, this may not be always the case, depending on which kind of protein/antibody/tissue you are looking at.

I just would like to state that neither quantifications in WB or IF are fully quantitative, but semi-quantitative, as you first need to obtain an image (be it from the microscope itself, or a band/blot/membrane), to analyze it pixel-wise (in terms of fluorescence, or in gray exposure density).

There are few REAL fully-quantitative protein quantification methods. Perhaps you should try an ELISA kit for the protein you are assessing? If can do that, at least you would have a third method to corroborate your results, and quantifications by ELISA are fully quantitative: you are going to put a concentration value to each case you are analyzing, something you could not do (at least properly) in WB or IF.

Hope this helps.