About the same time I got inspired to try making the vesicular protease assay, I figured I’d also build an assay to try to look at cell-surface localization of proteins.
Going back to that post about dL5 and malachite green (MG), I believe that MG doesn’t cross the the plasma membrane particularly well (Although now that I look at it, there are both ways to increase and decrease it’s cell permeability). Thus, I figured I’d test the dL5 fluorogen activating peptide in two forms; one where it’s sent out to the cell surface using a signal peptide (but anchored to the plasma membrane with a transmembrane domain), and another where its expressed as an intracellular, cytoplasmic protein.
Well, we made the constructs, and Sarah recombined the cells and tested them, and here are the results.
Well, ignore the blue distribution for now (since this is a construct testing a different hypothesis), and only look at the red and orange distributions for now. As you can tell, in the absence of MG, the signal is pretty low (I should probably through in some unrecombined landing pad cells on that plot to show what the background level is). On the other hand, MG addition causes cells encoding the extracellular dL5 to exhibit ~ 3e4 near infrared MFI, while the intracellular dL5 cells had about 1e4 MFI. So while that’s only a 3-fold difference, the standard deviations of those distributions were pretty tight, such that there was rather small overlap between those two distributions. So while this is a single, one-off experiment, it looks like this assay format may work.
Probably some additional knobs that can be turned to try to improve signal over background. First, is maybe this effect is somewhat MG concentration dependent, and reducing the amount of MG that is added may add some more dynamic range. Also, there are those less cell permeant MG derivations, which will likely improve the range (albeit, these are likely far harder to get than OG MG)