fluorescence quenching of tryptophan II

Frank Fürst ffrank at rz.uni-potsdam.de
Fri Jun 14 06:19:49 EST 2002


kerstin.janisch at bbsrc.ac.uk schrieb:

> Hello, 
> 
> thanks for the suggestions. I just want to know now, what purity some
> recommend for the testsystem.

That depends on the questions you want to answer. If you want to
determine binding constants of your ligand and/or the number of specific
binding sites for the ligand on your protein, you need a very pure
preparation in terms of protein contaminants, say better than
95%. Seeing no band on a coomassie stained gel might not be sufficient,
homogeneity on a silver stained gel is usually o.k. 

Additionally you have to make sure that the preparation is pure of the
ligand and any analogs of it that might have been present in the cell
from which the protein was purified.

Contamination by other, low-MW substances that do not interfere with
your assay are less important, as long as your signal-to-noise ratio is
good enough. 

If you just want to show qualitatively that your ligand interacts with
that protein, you don't need so high purity. Be aware, however, that a
contamination of 10% by a protein that gets quenched to 50% of its free
fluorescence upon binding of the ligand in question will give you 5%
signal change even if your protein doesn't interact at all. On the other
hand, the pure protein might not be quenched more than that even if it
strongly interacts with the same ligand, e.g. because the tryptophan
near the binding site is solvent exposed anyway.

Thus, if you have a major protein contaminant, you should make sure what
that is. Or you could use a different assay, like crosslinking or
enzymatic assay, if the protein is regulated by the ligand.

Bye, Frank
-- 
> But I don't really see running SETI at Home as practical as Folding at Home.  What,
> exactly, would be the benefit of finding intelligent aliens on the other side
> of the galaxy?
Maybe they're broadcasting the principles of protein folding... [from bionet.*]




More information about the Proteins mailing list