Jason Brinck (j.d.brinck at bham.ac.uk) wrote:
: Can anyone give me some info?
: I am at the moment purifying the recombant and wild type forms of an enzyme
: and want to analyse the differences in protein folding, if any, as the
: recombinant form is less active than the wild type. What type of analyses are
: there for studying protein folding and how much/how pure does the protein have
: to be. Circular dichroism has been suggested to me.
CD is good. You need mL's of uM protein soln, and it needs to be fairly
pure (as in > 95% ideally). I've heard FTIR is good for judging folded
states (as in 2nd structure content) but have never used it.I understand
its heavy on protein.
I'd recommend fluorescence, espc doing a fluoresence monitored
denaturation - compare wt and recombinant denaturations. Or you could just
look at the full emmision spectrum, but I think a denaturation would be easy
and probably be more convincing that it was folding ok.
Or, there's always NMR, if you've got access to one - just compare the
fingerprint regions of the wt and recombinant - don't even need them
assigned, judst overlay them (or even just 1D's). This has got to be the
most thorough, but its heavy on protein and time, and probably not what you
want to do first.
: Thanks,
: Jason Brinck
No problem.
Ben
--
______________________________________________________________________________
Ben Davis,
MRC Protein Function and Design,
Cambridge, UK
______________________________________________________________________________
"They can make me do it, but they can't make me do it with dignity."
