Ligand capture and Affinity Determination Methods

Antibodies or nanobodies can be used to immobilize proteins on various surfaces, such as microtiter plates, beads or biosensors. Protein-protein interactions and affinity (kon/koff and KD values) can be measured with ligand capture-based assay setups, e.g. by surface plasmon resonance (SPR, BiacoreTM/GE Healthcare), biolayer interferometry (BLI, FortéBio/Pall) or the switchSENSE® technology (Dynamic Biosensors). As a prerequisite for these assays, the affinity of the antibody or Nanobody to the capture ligand has to be high enough to assure ligand binding during the time of the measurement.

ChromoTek’s anti-GFP Nanobody has an extremely high affinity to GFP, with a KD value of approx. 1 pM, as determined by switchSENSE. Note that the other Nanobodies from ChromoTek like the GST, MBP VHHs do have single digit nanomolar KD values, which also allow for high affinity binding in before mentioned applications. Therefore, it is a perfect tool for capturing GFP-tagged proteins for protein-protein interaction studies and affinity determination. Furthermore, its high stability allows regeneration and reuse of the biosensor for multiple measurements.

Nanobodies as capture ligands

Nanobodies may be used as capture ligands because of their extraordinary chemical stability and high affinity; this approach may be also referred to as indirect ligand capture. In general, a very strong interaction between the capturing molecule, i.e. single domain antibody, and the ligand is needed to avoid ligand dissociation from the sensor surface, e.g. the ChromoTek GFP VHH binds to GFP derivatives with a very high affinity of just 1 pM.

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Antibodies as capture ligands

Antibodies may also be used as capture ligands since there are many antibodies commercially available. They do, however, need to have high affinities to avoid ligand dissociation from the sensor surface. Compared to Nanobodies, conventional antibodies are rather fragile. Thus, immobilized IgG may not be regenerated effectively and frequently.

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Looking for references? Please check our literature database.

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