ProteOn XPR36, User Manual

Page: 237 / 288

Determining the Optimum pH for Immobilization
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• Contact time — Increasing the contact time of ligand with the chip
surface increases immobilization levels up to the point that
saturation is reached, or the intermediate active ester groups have
decayed away. Limit contact time to what is necessary to preserve
ligand samples. For analysis of protein-protein interaction kinetics,
a contact time of 1–2 min is recommended. Longer contact times
of 5–6 min are recommended in cases where higher immobilization
levels are required, such as analysis of small-molecule interaction
kinetics and concentration determination assays
Determining the Optimum pH for
Immobilization
Use an inactivated sensor chip to study the electrostatic attraction of ligand to
the sensor chip surface.
To determine the optimum pH for immobilization:
1. Prepare 100 μl of ligand at 20 – 50 μg/ml in pH 5.5, 5.0, 4.5, and 4.0
buffers. A pH of 6.8 is optimal for sulfo-NHS.
2. Inject the samples (typically at 30 μl/min), and observe the binding
response curves (adsorption SPR sensorgram).
3. Use the highest pH at which saturation of protein adsorption occurs on
the sensor chip surface.
4. After the injection, confirm that most unbound protein dissociates after
washing with running buffers. Ligand bound electrostatically to the chip
surface should dissociate when injection of running buffer resumes
because its net positive charge decreases in higher pH. Also,
electrostatic interactions are screened by the higher ionic strength of
the buffer. Minor amounts of protein may remain attached. Remove this
protein by injecting the deactivation buffer, which has an even higher
pH and ionic strength. Additional solutions that may be useful for this
purpose include regeneration solutions of 50 mM NaOH, 1 M NaCl, or
any mixture of the two.