When I am confronted with a frustrating problem, I like to run a “sanity check” to test my assumptions. For instance: I’m running this FP assay. When I add protein, I should get a change in the fluorescence polarization in my sample. There’s lots of literature that suggests it does happen.But is my instrument capable of detecting it?
As a sanity check, I ran control samples in my plate reader 36 times. I took the plate out, put the plate back in, and ran the same samples in my plate reader 36 times. I need to know about my reader’s stability.
I expect a relatively small ΔFP. The fluorescein is attached to a 20 kD DNA aptamer which binds to a 16 kD protein. That’s not a very big change in molecular weight. The previous binding assay gave a max ΔFP of 10 mP. I need a standard deviation of less than 3 mP to have any confidence this measurement.
Thankfully, the standard deviation of the ΔFP across 22 wells was 2 mP. The well-to-well-variability was terrible. Each well needs its own control.I can’t measure absolute FP with any accuracy, but I should be able to measure ΔFP if it’s greater than 6 mP. That gives me more confidence about the measurements from the last few days.
I looked for some literature to compare. A small fluorescein-labeled peptide (MW 1.3kD) binding to a large fusion protein (49 kD) gives a ΔFP of ~200 mP. The scatter around their curve is about 6 mP.That’s similar to my own. If I get a ΔFP of that magnitude, I should be able to measure it easily. I measured the FP of erythrosin and got ~340 mP so I am definitely able to detect FP when it’s strong (literature value 316 mP).