I need some help, because I have silenced a protein using lentiviral transduction particles, at least when I perform a western blot the protein is not there, but when I perform an immunofluorescence assay I can see it, actually I'm not sure if its just an inspecific signal or I'm doing something wrong. I fix the cells using PFA 4% in PBS and I permeabilize them using Triton 0.1%. I think it is not a problem with the secondary antibody, because I've already checked it. Can someone help me?
Thanks in advance
I think it is easier to see a difference of amount in a WB than IF, mainly because in WB, the visualization of the protein of both situation is performed on the same membrane, at the same time. But, perhaps, if you increase protein amount of both treated and control you could detect a slight amount of protein in treated cells. And a large amount in control cells....
In IF, it is 2 different visualizations and due to an high sensitivity a low amount of protein might be detected.
Hi Ana! so..first..you cannot never compare expression levels between WB and IF, because of the different methods used. Even if the primary Ab is the same, the secondary is different and so the avidity of the secondary is different. Moreover, quantitate IF is a tricky process so..forget about that :)
what you can do is check for the specificity of your 1 Ab..either using a KO or a tissue where your protein is not expressed, or making serial dilution...i.e both treated and untreated cells with serial dilution of 1 Ab, and check when the signal disappear in your silenced sample, and check if it does not disappear in untreated sample...
I suppose you have done the appropriate controls for IF: no 1 Ab, no 2 Ab, no 1 and 2 Ab..
good luck!
Try staining with the secondary antibody alone to check aspecific signal.
Thanks very much for all your suggestions, I'll try some of them
Make sure you have 1st antibodies reactive to your target proteins,either native or SDS-denatured form. If so, you should be able to check the feasibilty of each of your 2nd antibodies used for IF and WB. Otherwise, did you try with readily known-controls,whether they can work properly.
Usually,each testing methods need to be pre-optimized. Then, you can determine the test results.
Do you see a difference in the fluorescence intensity between the cell lines? If you do then you might not have completely shut down the expression level.
If you load more amount of your samples for WB, you might start detecting the expression of your protein in shutdown cell lines.
WB is more conclusive than IF for conforming and more quantifiable.
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