Biacore -Reply

Tom Haas Ph.D. (haast@cesmtp.ccf.org)
Thu, 03 Dec 1998 09:23:30 -0500

I have used numerous detergents on the Biacore 1000 with great results.
These detergents include:Triton X100, Nonidet P-40, Nonidet P-20,
Tween 20 and octaglucoside. I find that the most important precaution to
take in order to get a stable baseline is to degass the buffers
extensively. I do this before adding the detergents to prevent foaming. I
have used them at concentrations ranging from 0.1% to 0.005%. I don't
think that glycerol will be a problem as long as the viscosity of the buffer
isn't to high that when the syringe sucks up the buffer that air bubbles
form. Give your Biacore Rep a call. I'm sure that they will know
someone who uses glycerol.

Tom Haas
Molecular Cardiology
Cleveland Clinic Foundation

>>> Deb McMillen <mcmillen@morel.uoregon.edu> 12/02/98 06:17pm >>>
Hi, all,

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