Flow Cytometry vs Western Blotting: can't get the same result. Why? - (Oct/16/2016 )

Hello everyone,

I performed WB and Flow Cytometry analysis of 2 cytoplasmic proteins in stable transfected HEK293 cells. All primary antibodies were monoclonal and already tested for Immunofluorescence.

The problem is:

WB showed, as we expected, a huge protein expression for protein A after induction with Dox and no band in my non-inducible cell. But for protein B, I got the same weak band in Dox (strange) and non-Dox treated cells.

However, flow cytometry (same primary antibodies) analysis showed:

    1) Decreased of Protein A and B after Dox treatment, including in my HEK control cells;

    2) In non-Dox treated cells, we saw an increase of over 50% of protein A and B expression!! 

    3) Unlike WB results, HEK control cells had a lot of Protein A and B;

I am veeeery confused right now.

Any ideas about what might be happening?

Thank you in advance!

Sounds like your antibodies detect the denatured form better than the native form; this is not uncommon.You may need to look at other antibodies for the proteins for use in FACS or test different conditions for these proteins.

Your first two statements are a little unclear 1) decreased A and B compared to what? other cells in the same experiment or relative to the WB? 2) Increase relative to what?