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RE: Better Functional Understanding From Studying Human Vs Neanderthal Enzymes
Fluorescence of proteins, my first love <3 and M.Sc. thesis, although I never used SYPRO Orange (shame on me)
Fluorescence of proteins, my first love <3 and M.Sc. thesis, although I never used SYPRO Orange (shame on me)
Sypro isn't a protein fluorescent tag. Its just a free dye which has a propensity to bind to anything hydrophobic. I can stick any greasy thing in the buffer and the fluorescence will spike. In my work, I deal with a lot of small molecules, and some of which can cause some really wonky data due to background interactions between the dye and the compound. Obscuring the protein unfolding that I am interested in.
We liked doing it Raw :D Tryptophan only
Those were very boring experiments:
Now I'm returning back to fluorescence and fancy probes for STED
When you use internal fluorescence you typically need to use a lot of protein 10-20 uM. However with sypro you can get very nice unfolding curves, usually with very minimal purturbation of intrinsic unfolding Tm (based on Trp fluorescence), at concentrations in the 1 - 2 uM range, and I have gotten down to as low as 250 nM for a few proteins. Depends on the amount of hydrophobic surface.
These are still boring experiments, I typically multiplex and do things in 384 well plate using a Q-PCR machine. So you can generate a whole bunch of data all at once.
Another fun little path is to use the 'proteoplex' software to quantitate and optimize unfolding conditions, really speeds up assay design. (http://www.proteoplex.de/)