I have a (complex) question about using glycerol and detergent in the
running buffer on a Biacore. Has anyone used glycerol (not just in the
samples being injected through the loop) but in the eluting buffer? How
much glycerol and what is the end result? Because it has a large
refractive index, is it largely that you have too high of an RU baseline
to see other interactions with sensitivity over this baseline? Does use of
the glycerol result in a large increase in noise?
We have a
Biacore X--is the sheer viscosity of the solution also a problem?
We are thinking of using a detergent other than Biacore's surfactant
P-20 for an experiment to keep
conditions in compatible with some other work that we are doing. Has
anyone used Nonidet P-40? This has a fairly high OD at 254 nm (0.29 for a
0.05% solution)?
Also, I would think it would be important to keep the detergent
concentration under the CMC of the detergent
so that large micellar complexes aren't formed with the analyte (if
that is possible) so that you can look at the kinetics of the analyte
interacting with the ligand and not, instead, first kinetics of the
analyte dissociating itself from a detergent micelle and then associating
with the ligand. OR having an effectively lower concentration of free
analyte to interact with ligand as some of the analyte is tied up in
micelles.
Any insight to experience with detergents/glycerol in the Biacore is
welcome.
Thanks, Deb McMillen
Institut of Molecular Biology
University of Oregon
Eugene OR