ProteOn XPR36, User Manual

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ProteOn XPR36 System | C Surface Plasmon Resonance
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(otherwise, dissociation of the ligand from the capture antibody
may interfere with interaction analysis)
• NTA attachment —uses an NTA coated surface (for example, HTG
chip) to capture HIS tagged biomolecules directly from crude
samples.
Activation Step (Amine Coupling)
ProteOn GLC, GLM, and GLH sensor chips are used for amine coupling,
which requires activation of the carboxylic groups on the chip surface. The
ProteOn NLC sensor chip, which is precoated with NeutrAvidin, does not
require activation.
Amine coupling uses a combination of activating agents, which must be
stable for the duration of the ligand immobilization process, and must
generate an effective leaving group to be replaced by the ligand. In the
ProteOn system, this is achieved by using a mixture of
1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDAC or
EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). These reagents form esters
that interact with amine groups on the ligand.
• EDAC — activates carboxylic groups easily, and because it
generates a good leaving group, it also detaches rapidly
• Sulfo-NHS — assists coupling in the presence of EDAC. The
reaction includes formation of an intermediate active ester (the
product of condensation of the carboxylic group and sulfo-NHS)
that further reacts with the amine group ion on the ligand to yield
the amide bond. Sulfo-NHS maintains a full negative charge on the
chip surface after activation, enabling more efficient coupling in
comparison to the EDAC-NHS reagent pair
Immobilization Step
During this step, ligand is injected across the sensor chip surface, which in the
case of amine coupling, is negatively charged. The negatively charged surface
attracts positively charged amine groups in the ligand, which then react
spontaneously with the reactive sulfo-NHS ester groups to form stable,
covalent amide bonds. In the case of biotin and HIS tag attachments, the
ligand contains a biotin or HIS label that recognizes and binds
noncovalently—but with very high affinity—to the NeutrAvidin or NTA moieties
on the chip surface.