The same primary antibody (that detects only the phosphorylated residue and not the non-phosphorylated form) was used in both immunoflouresence and western blot. When treated with kinase inhibitor, phosphorylation is pretty much eliminated on western blot while controls still have phosphorylation.
When I treated the same cells with the same concentration and same time followed by immunoflouresence instead of western blot, I can still see staining of the phosphorylated protein.
Any suggestions for the conflicting results?
First thing which comes into my mind is unspecific binding of the antibody in immunoflouresence. Even if it's the same antibody, it can act quite differently in WB vs IF...
First thing which comes into my mind is unspecific binding of the antibody in immunoflouresence. Even if it's the same antibody, it can act quite differently in WB vs IF...
the IF signal may come from something else than your target P-site - a lot of antibodies work only partially well for IF
You have done exactly the right experiment to show that this particular antibody is not suitable for IF. In our hands, even if an antibody is listed as good for IF, we have a 60-70% attrition rate when we test against western blotting.you can try to optimize the IF experiment, but likely you will need a different antibody or run a different experiment.
I would use a negative control to make sure it is not autofluorescence or nonspecific binding.
Sounds like non-specific IF staining is the problem here. The specificity of the immunoblot is enhanced because you separate all cell proteins by SDS-PAGE. However, there is obviously no such separation with IF staining of cells where proteins are fixed. Hence, even a small amount of background non-specific staining can add up to a failure to observe the result predicted by the immunoblot analysis in the immunofluorescence analysis. There is not much that you can do except to further purify your p-specific Ab to try to remove non-specific staining or try another antisera that is p-specific for your protein.
Separation is certainly one reason why WB is more likely to work than IF. But other things factor in as well. For example the presentation and accessibility of the antigens is much better on WB to promote antibody binding, while on the other hand fixation and permeabilization during IF can alter antigens to the point that they are not recognized by the antibody. I think it is not certain that even if all non-specific binding was abolished the antibody would work for IF.
This is a frequent problem with phosphor-specific antibodies which can have residual, albeit slight avidity to the unphosphorylated epitope. Since the local concentrations of the antigen may be very high in some areas of the cell, the antibody may have difficulty in discriminating the non-phospho-epitope by IF. Secondly, the proteins' stability may be regulated by phosphorylation. The kinase inhibitor may therefore increase the abundance of the protein, making it more easily detectable compared to the phosphorylation protein. Although this should also be apparent by immunoblotting, as has been said above, there is not a linear correlation between IF and immunoblot modes of detection, neither of which are particularly quantitative, with IF being virtually qualitative.
Are doing direct or indirect IF? Have you tried to reduce the amount of antibody (primary or secondary if doing indirect) in the IF experiment? Do you have a control for the secondary antibody if doing indirect to make sure there is no non-specifc binding? Also, how are you fixing the cells, if you are using formaldehyde are you making it up fresh?
I agree with the previous comments that virtually IF may have higher nonspecific binding in the background but there are opportunities to reduce the back ground by testing different washing buffers. One thing, I wonder if you have been able to detect phosphorylation by WB in control cells or other cell lysis? by these a.b.? If you have then it is not strange, since the inhibition of kinases reduces phosphorylation level , may be down to 5-10% of the total level. If you have not been able to detect phosphorylation using these antibodies then it might mean that they do not work for wb. Or the level of phosphorylated proteins are so low that you can not detect with wb. Normally IF can be slightly more sensitive than WB but there are ways to improve your WB .. Which detection reagents do you use? ECL Slect. Prime or advans ..? One more thing you can improve your WB with Signal Booster Buffer (from Beacle, Japan) Enhancing the signal detections with 10 to 100 fold or more without Affecting the background level. ..Here you dilute your a.b in this buffer and you just continue your wb protocol.
Good luck
Yes, I can detect phosphorylation in untreated controls on WB so the antibody is fine is WB.
I also tried a negative control with no primary antibody but with 2ndary (alexa fluor 568) and there's no non-specific binding/autoflourescence.
I'm not sure about non-specific binding of the primary antibody. Since phosphorylation of my protein only occurs in S/G2 cells and the fluorescence I see is limited to these cells even in treated samples.
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