ProteOn XPR36, User Manual

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Ligand Immobilization Process
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For analyte interaction, fluid containing the analyte flows through the channels
in the second (orthogonal) direction, creating the 6 x 6 interaction array. The
association phase of the interaction is monitored in real time. Running buffer is
then pumped through the analyte channels, and dissociation of the analyte
from the ligand is monitored in real time. Following this analyte phase, the
immobilized-ligand surface may be regenerated.
Ligand Immobilization Process
Immobilization Chemistries
A number of methods and chemistries are available for the immobilization of
proteins (and other biomolecules) to the sensor chip surface. The type of
ligand used usually determines the type of sensor chip (surface chemistry)
and the type of immobilization chemistry used. The most commonly used
ligand immobilization chemistries are the following:
D
IRECT
, C
OVALENT
C
OUPLING
Direct, covalent coupling uses functional groups in the ligand to form covalent
bonds with carboxylic functional groups on the sensor chip surface. This
method requires no ligand modification, but usually results in a random
orientation of the ligand, which may limit the accessibility of the binding site.
Immobilization levels are generally high, and ligand consumption is generally
low using this approach.
Amine coupling uses primary and secondary amine groups in the ligand for
coupling with carboxy-coated sensor chips. The carboxylic groups on the
sensor chip surface (for example, ProteOn GLC, GLH, and GLM chips) form
amide bonds with the ligand.
N
ONCOVALENT
C
OUPLING
Noncovalent affinity coupling allows oriented attachment of ligand molecules
to the sensor chip surface. Examples of this type of coupling include:
• Biotin attachment — uses an avidin-coated surface (for example,
the ProteOn NLC sensor chip coated with NeutrAvidin) and
biotinylated ligand molecules, which often require purification prior
to analysis. For interaction analysis, the biotin label must not
interfere with the ligand binding site
• Antibody capture — uses a layer of antibodies (immobilized by
either covalent or noncovalent coupling) to capture the ligand of
interest. The antibodies must have a strong affinity for the ligand