Biacore X100 reference portal

Surface thiol coupling relies on thiol-disulfide exchange between a thiol group on the dextran matrix on the sensor chip and a reactive disulfide introduced into the ligand.

Chemistry

The thiol group is introduced to the surface via amine coupling of cystamine followed by reduction with DTE. The disulfide group can be introduced to the ligand in a separate step through reaction with 2-(2-pyridinyldithio)ethaneamine (PDEA) or other commercial reagents.

Excess reactive groups are deactivated with PDEA/NaCl. Surface thiol coupling should be used with care if the analyte requires reducing conditions.

Surface thiol coupling

The surface thiol approach can be applied to any ligand into which a reactive disulfide or maleimide group is introduced. Examples include modification of amine groups with heterobifunctional reagents that are activated with NHS, such as N-succinimidyl-3-[2-pyridyldithio]-propionate (SPDP) and 4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]-toluene (SMPT), and modification of aldehydes (created by oxidation of cis-diols) with N [ε maleimidocaproic acid]hydrazide (EMCH).

Immobilization procedure overview

The general pattern of steps involved in immobilizing ligand is the same for essentially all covalent immobilization methods:

• Activate the surface by injection of appropriate reagents.
• Inject the ligand solution.
• Inject reagent to deactivate remaining active groups on the surface and remove non-covalently bound ligand.

There may be additional steps depending on the properties of the ligand, the type of sensor surface and the details of the chemistry used.

Lab protocol

Lab procedure Surface thiol coupling of ligand to Biacore sensor chips is available for download on www.cytivalifesciences.com/solutions/protein-research/Interaction-analysis-with-Biacore-surface-plasmon-resonance-SPR/Get-started-with-surface-plasmon-resonance-SPR-interaction-analysis