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RE: Better Functional Understanding From Studying Human Vs Neanderthal Enzymes
We liked doing it Raw :D Tryptophan only
Those were very boring experiments:
- take the protein
- put it into the fluorimeter
- turn the heating on
- measure it
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/)