ChromoTek’s unconjugated Nanobodies or VHHs are well characterized and validated for the listed applications. Because the molecular weight of these single-domain antibodies (sdAb) is just about 15 kDa, VHHs are only one tenth the size of a conventional IgG antibody (150 kDa) or less than a third the size of a Fab fragment (50 kDa). In combination with their high chemical and thermal stability plus high affinity, these binding proteins are ideal for multiple types of affinity assays in biochemistry and microscopy, including immunoprecipitation, immunofluorescence, super resolution microscopy, surface plasmon resonance (SPR), biolayer interferometry (BLI), switchSENSE technology, etc.
GFP VHH, biotinylated
p53 C-term VHH
p53 N-term VHH
Ligand capture and Affinity Determination Methods
Label-free methods for the measurement and characterization of biomolecular interactions are very popular and widely used, e.g. when conducting binding assays and screenings. Here, on-rates, off-rates, and affinities are determined based on an experimental set-up, in which a ligand is captured to a sensor’s surface; the ligand then interacts with an analyte that the sensor is probed with. Applied assay technologies comprise surface plasmon resonance (SPR), e.g. from BiacoreTM/Cytiva (formerly GE Healthcare), bio-layer interferometry (BLI), e.g. from FortéBio/Sartorius, and switchSENSE® from Dynamic Biosensors.
The stable, uniform and oriented, i.e. site-directed, immobilization of the ligand is of considerable importance for the reliable determination of the binding kinetics of ligand to analyte.
In general, these assays are distinguished by the immobilization of the ligand to the sensor’s surface:
- Direct immobilization assays:
Covalent linking via lysines or cysteines of the ligand and click chemistry
- Indirect immobilization assays or capture kinetics assays:
Capturing the ligand via specific binding molecules like antibodies or Nanobodies in a site-directed, i.e. Fc-domain specific manner or via antibodies or Nanobodies against protein- or peptide-tags like GFP, GST, MBP, Spot-tag, His-tag, etc.
Common capture molecules for antibodies are anti-Fc specific antibodies and Nanobodies like Nano-CaptureLigands™, or protein A, protein G, protein A/G.
Compared to direct immobilization assays, the benefits of capture kinetics assays are
- site-directed immobilization,
- regeneration is a standard procedure and does not depend on the captured molecule, because the ligand is removed after every cycle,
- the ability to capture unpurified monoclonal antibodies from crude samples,
- easier optimization of capture ligands’ density.
Only for research applications, not for diagnostic or therapeutic use!