Using Mass Spec to Detect the Barcikowski Protein
This week I tried looking into several different ways of detecting the elusive Barcikowski Peptide. We discovered that Coomassie staining does not work, both because of the size of the peptide (it only works on proteins with a MW > 3000Da) and because Coomassie preferentially binds basic residues (and with a sequence of CEEEEEEEEEEEEW, we’re just about as acidic as you can get). So I got in touch with Fred Hagen from the proteomics center. He’s quite confident that Mass Spec can be used to quantify the protein. Mass spec is a pretty big hammer for the small nail of just detecting this protein, but the Barcikowski group has told me they are actually interested in the mass spec results anyways.
More generally, I’d like to find someone who has a better UV-Vis spectrometer that can detect proteins at this concentration. The Barcikowski lab is using a Thermo Scientific Evolution 201 to generate beautiful curves at 20uM peptide. Ultimately I think our TECAN was designed for DNA and RNA detection, and while we can usually detect protein with it, at low concentrations it is calibrated for other things (especially the nanoquant plate that we’re using).
I’ve used a Perkin Elmer UV-VIS spectrometer in Prof. Krauss’s lab on the 2nd floor of Hutchinson. I don’t know if the sensitivity is better or worse than the Tecan, but it might be worth investigating.
Looking at the Thermo tool, I can’t see how it is any better than the Tecan. Are you using it exactly the same way as the B’ski lab? Are you using a high quality quartz cuvette with the longest path length possible? 20uL of a tiny peptide is not a lot. Have you looked at all the possible absorption peaks? 280, 250, 219 nm? You mention the nanoquant plate – this substantially limits sensitivity, correct? It must if the path length is extremely short. If you are volume limited, you may be better off diluting it and using a good qz cuvette.
Anyway if the other guys are not lying or confused, you should be able to repeat their data on the Tecan if it is working properly. Good luck.