ProteOn XPR36, User Manual

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Ligand Immobilization Process
217
The extent of ligand immobilization determines the binding capacity for the
analyte. It should be high enough to allow sufficient response to the analyte,
but not so high as to generate crowding effects or limit mass transport. The
standard analyte response that gives the best data is 100–200 RU. The ratio of
the mass of the ligand to the mass of the analyte can be used to determine
how much ligand should be immobilized to produce a specific analyte
response. An easy way to determine optimum surface density is to calculate
the theoretical R
max
of the interaction you are studying. The theoretical R
max
is
the maximum analyte response assuming that the ligand is 100% pure and
100% active and that all binding sites are available:
R
max
is the maximum theoretical response of the analyte for a given
ligand level
L is ligand
A is analyte
R
L
is the amount of ligand immobilizied
MW is molecular weight
n is the stoichiometry of the reaction
The extent of ligand immobilization depends on the following factors:
• Buffer selection — for amine coupling, the ligand is immobilized in
a buffer that ensures a net positive charge on the ligand, so that it
is attracted to the negatively charged chip surface. Thus, the buffer
must be of low ionic strength to minimize charge screening.
However, amine coupling is most efficient at high pH, because
activated carboxylic groups react better with uncharged amino
groups. The highest pH compatible with preconcentration is
determined empirically, as described below. For amine coupling,
buffers should not contain free amine or nucleophilic groups (for
example, Tris) that can react with the activated chip surface.
Sodium acetate buffers with pH 4.0
– 5.5 are generally used for
amine coupling. For biotin attachment, ligand is generally dissolved
in running buffer
• Activation level of the sensor chip surface — the extent of
activation influences the efficiency of immobilization, and may even
be varied in a controlled manner for certain applications (for
example, one-shot kinetics). The concentration, flow rate, and
injection times for the activating agents all influence the extent of
activation. ProteOn GLC sensor chips have easily activated
carboxylic groups and a binding capacity of approximately one
protein monolayer, while GLM sensor chips have easily activated
R
max
MW
A
MW
L
------------
R
L
×
n
×
=