Answers to Frequently Asked Questions When Preparing For A
BIA Assay
I. BUFFERS
Primary amine buffers must be avoided for amine
coupling. It is important that there are no competing amine
groups in the buffer (i.e. Tris or glycine, BSA, etc). For
example,Tris and TBS buffers cannot be used due to the
excess binding of free amine groups in the immobilization
procedure.
HEPES is strongly recommended as a buffer at 10mM HEPES, pH
7.4 / 150mM NaCl.
Buffers containing glycerol or sucrose cannot be
used. These components may alter data due to their elevated
refractive indices.
The salt content of buffer should be < 200 mM.
A. Running Buffers
An NaCl concentration of 150 mM or greater is
helpful in reducing charge effects.
All buffers should be filtered with 0.2 m M filter
and thoroughly degassed.
Low levels of nonionic detergent (i.e. 0.005% P-20
or Tween-20) should be incorporated.
II. ANALYTE - the molecule to be passed across a chip
immobilized ligand.
Some measure of purity should be provided.
Purity may be evaluated by SDS-PAGE and Coomassie
staining.
The BIAcore staff may request such verification.
For kinetic studies, purity should be > 90%
It is important that the molar concentration of
the analyte is known. This will ensure an accurate kinetic
analysis.
For kinetic studies, the molecular weight of the
analyte should be 2kD or greater.
The concentration should be 0.1 -1 mg/ml, if
possible (total consumption depends on affinity,
typically 10-100ug).
If glycerol is present in samples it should be
less than 5%.
The exact concentration and molecular weight are
desirable in interpreting results.
Proteins with pIs greater than 9.0 show a high
degree of non specific binding to carboxydextran
surfaces.
III. LIGAND - the molecule to be coupled to the surface
of the sensor chip.
Most proteins are readily coupled though free
amines (lysines or amino-terminus). There are, however,
other coupling chemistries available, including thiol and
aldehyde. There are also capturing schemes that employ
biotin, GST, 6xHis, or antibodies.
Ligand concentrations should be between 0.5 - 5
mg/ml (sample consumption ~10ug total).
It is also necessary to know the approximate
molecular weight and pI of the molecule.
