Hi,

I have a question about FACS and copy number estimation of a protein of interest.

We have a protein of interest (on the membrane surface) that we are studying and we have an antibody coupled with PE that we use for FACS analyses. It works great and we don't have issue with the antibody. We determined the copy number of our protein in multiple cancer cell lines by using in parallel the Quantibrite PE beads from BD Biosciences (they are calibrated beads, all uniform, with four different amount of PE payload, giving you 4 different peaks with each peak corresponding to a number of PE associated molecule. You can then determine proportionally your copy number for your protein of interest).

On different positive cell lines, we have a nice shift of PE intensity between the isotype control and the cells tested with our antibody. When we did the same experiment on tumor samples (so mixed with different populations of cells), we also obtained a nice shift between the isotype and the tested sample (at least 3 Log10 fold of difference). Our tumor samples are previously blocked with Fc-blocker and we use a saturating concentration of antibody.

But when we run the beads for the tumor samples, the peaks shift to the right compared to when we analyze cancer cell lines. The consequence of it is a drastic decrease estimation of copy number. The voltage for SSC, FSC and PE can variate between the two, but the difference between the isotype and the antibody of interest is similar, so why do we get this shift with the beads but not with the isotype?

The confusing part for me is we get, let's say, 150K copy for the cell line but only 5K copy for the tumor, but the log10 difference in intensity (between isotype and test) for the two condition is similar (not a small shift for the tumor sample). I can't explain it. If you have any idead, that would be much appreciated, thank you!