Learn Biacore from Publications
The mAbs 127 and 150 specifically bind to SARS-CoV-2 S1-RBD. (A) Affinity of mAbs 127 and 150 for S1-RBD. The S1-RBD-His-bound sensors were incubated with the different concentrations of mAbs 127 or 150 (indicated by different colors) for a set time interval to allow association. The sensors were then moved to protein-free solution and allowed to dissociate over a time interval. The binding kinetics were determined using Biacore T200 Evaluation Software version 3 with 1:1 binding model fitting. (Publication title as below and link)
Lai YC, et al. . Antigenic cross-reactivity between SARS-CoV-2 S1-RBD and its receptor ACE2. Front Immunol (2022) 13:868724. doi: 10.3389/fimmu.2022.868724 - DOI - PMC - PubMed
Biacore Assays details from the publication
Why you shouldn't immobilize His-tagged proteins directly on sensorchip by Amine coupling methods
- They can result in random immobilization of the histidine-tagged proteins on the sensorchip surface, which can affect the orientation and accessibility of the His-tag.
- They can lead to non-specific binding and reduced binding activity of the protein. Such as in this publication, the calculated Rmax is about 4144 RU, but the binding RU showed is too low to be believable.
- They can potentially block the His-tag site of the protein, leading to reduced or no binding activity.
Methods for immobilization of Histidine-tagged Proteins
The best way to immobilize a His-tagged protein for Biacore analysis depends on several factors, such as the specific experimental setup, the properties of the protein, and the ligand of interest. However, here are a few commonly used methods for immobilization:
- Ni2+-based Immobilization: This method involves immobilizing the His-tagged protein onto a sensorchip pre-coated with nickel ions. The His-tag of the protein binds to the nickel ions, allowing the protein to be immobilized.
- Antibody-based Immobilization: This method involves immobilizing an anti-His-tag antibody onto the sensorchip, which can then bind the His-tagged protein. This approach is particularly useful when the protein of interest is difficult to immobilize directly or when multiple proteins need to be immobilized simultaneously.
- Streptavidin-based Immobilization: This method involves immobilizing a biotinylated His-tagged protein onto a sensor chip pre-coated with streptavidin. The biotin binds to the streptavidin, allowing the protein to be immobilized.
- Direct Immobilization: This method involves immobilizing the His-tagged protein directly onto the surface of the sensorchip. This can be achieved by amine coupling or thiol coupling, depending on the functional groups available on the protein.
Each of these methods has advantages and disadvantages, and the optimal method may depend on several factors, including the binding kinetics, the specificity of the interaction, and the stability of the immobilized protein. It is important to perform a preliminary experiment to determine the best immobilization method for the specific experimental setup.
Biacore and SPR technology in Applications