Biacore X100 reference portal

Immobilization of macromolecular ligands is usually performed from reasonably dilute ligand solutions (10–50 µg/ml or less), and result in immobilization levels that correspond to a concentration in the dextran matrix of the order of 10–20 mg/ml or more ¹. This is achieved through electrostatic pre-concentration of ligands in the dextran matrix.

At pH values above about 3.5, the carboxymethylated dextran on the sensor chip surface is negatively charged, and electrostatic attraction provides an efficient means for concentrating positively charged ligands on the surface. This mechanism works well for most proteins and for several other kinds of biomolecules.

The primary requirement for this electrostatic pre-concentration on the surface is that the pH of the ligand solution should lie between 3.5 and the isoelectric point of the ligand, so that the surface and the ligand carry opposite net charges. In addition, the electrostatic interactions involved in pre-concentration are favored by low ionic strength in the coupling buffer. In general, covalent immobilization of proteins is typically performed from solutions in 10 mM buffer (e.g. sodium acetate) at pH 4–5.5. A range of ready-to-use buffers for protein immobilization is available from Biacore. The extent of pre-concentration of ligand on an unactivated sensor chip under different buffer conditions can be measured to determine the suitable conditions of pH for immobilization.

Ligand is concentrated on the surface through electrostatic attraction when the pH lies between the isoelectric point of the ligand and the pKa of the surface. If the pH is too low or too high, ligand will not be concentrated on the surface.

Some ligands (for example highly acidic proteins and nucleic acids) are negatively charged even at low pH values and cannot be efficiently pre-concentrated on carboxymethyl dextran surfaces. Modification of the ligand can in some cases help to reduce the acidity: for example introduction of active disulfides for thiol coupling may raise the isoelectric point of proteins sufficiently to allow pre-concentration. High affinity capture is an alternative immobilization approach that does not depend on electrostatic pre-concentration: attachment of biotinylated nucleic acids to streptavidin-coated surfaces is one example. Capturing interactions can be exploited in buffers of physiological ionic strength or higher to reduce the effects of electrostatic repulsion of acidic ligands.

¹ An SPR response of 1,000 RU corresponds approximately to a surface concentration of 1 ng/mm2 for an average protein ligand on Sensor Chip CM5. If the thickness of the dextran matrix is taken to be 100 nm, this is equivalent to a volume concentration in the dextran matrix of 10 mg/ml.

For many proteins, coupling in 10 mM acetate buffer pH 5 works well. If you need to use other conditions, bear the following considerations in mind:

• The buffer pH should be at least 0.5–1 unit below the isoelectric point of the ligand. For ligands with isoelectric point above pH 7, the buffer pH may be increased to 5.5 or 6. The optimum pH can be determined experimentally.
• The ionic strength should be low (recommended 10–20 mM monovalent cations).
• Buffer components containing primary amine groups and other strong nucleophilic groups (e.g. Tris, sodium azide) must be avoided for amine coupling, since these will compete with the ligand for activated esters on the sensor chip surface. Thiol coupling must be performed in the absence of reducing agents.