>Hi, all,
>
>Has anyone compared use of Neutravidin to Streptavidin in making your own
>SA chip surface for experiments in the Biacore? I like the idea of being
>able to create a surface that is less dense than the SA chip that Biacore
>sells--and coupling your own avidin species is a lot less expensive. The
>Neutravidin seems to also be better to use as some hydrophobic patches and
>charged regions that are on the surface of Streptavidin have been
>engineered "out".
>
>Deb McMillen
>Institute of MOlecular Biology
>University of Oregon
>Eugene OR
Hi Deb,
For the last four years, I have been making my own biosensor chips with
Neutravidin with great results. I find that Neutravidin has lower
nonspecific binding than streptavidin plus it is cheaper to make your own
chips than purchasing SA chips from Biacore. Here's what I do: Dissolve
Neutravidin (Pierce Chemical) in HBS at 1 mg/ml. Dilute 1:20 with 10 mM
acetate, pH 5.0, then perform the standard EDC/NHS coupling protocol
suggested by Biacore. After this you have to stabilize the baseline by
regenerating the chip with 0.1% SDS. Make three injections, 2' contact
time, over the surface. To couple biotinylated peptides, I prepare a 50nM
solution of peptide. For immobilization, set the flow rate to 10 uL min-1
and inject 5uL for a 30 second contact time. These injection conditions
will produce a biotinylated peptide surface density of ~50 RU. You must
watch the relative response as the ligand is immobilized on the chip. It is
very important that no more than 25 to 50 RUs of biotinylated peptide is
immobilized if you are considering kinetic analysis. Therefore you must
watch the relative response and stop the injection when >50RUs are bound.
You can control (reduce) the amount captured on the surface by a lowering
the peptide concentration, shortening the contact time, increasing the flow
rate or any combination of these. Vice versa if you want to capture a large
RU level for equilibrium binding analysis or ligand fishing experiments. I
typically use 0.05% to regenerate the surface when performing interaction
analysis.
Good luck with your experiments.
Chris
Weather Inconsequential. I drove to work today in a massive downpour. Of
course, this is unusual for Southern California. Now, an hour after I
arrive, the sun is burning brightly through the clouds. The folks up north
in LA even had a tornado. Now THAT is definitely not inconsequential.
***********************************
Christian R. Lombardo, PhD
Director, Molecular Analysis Facility
The Burnham Institute
10901 North Torrey Pines Road
La Jolla, CA 92037
858-646-3108
crl@burnham-inst.org
***********************************
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