Hello,

I am trying to assess the rate of protein translation in two different cell samples: a WT and a KO. I am using OP-Puro incorporation as the assay of choice and I am looking at the fluorescence signal on FACS after click-chemistry reaction as my readout.

My problem is that my WT cells also express GFP, while KO cells lose this signal (the gene we KO'ed is fused to GFP). Thus, after acquisition on FACS, all my WT cells show both GFP and OPP antibody signal, while the KO cells only show the OPP antibody signal. I have tried to compensate for the bleedthrough between GFP and the OPP antibody for the WT samples, but I am still unsure whether my readout for the OPP median fluorescence intensity between the WT and KO in this setting is indeed comparable and accurate.

Do you have any suggestions on how to make sure I only calculate the OPP antibody signal in both WT and KO cells, without artificially reducing WT values (by over-compensating to account for GFP) or artificially increasing WT values (by including GFP signal in my OPP antibody readout)?

Thanks a lot!