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Capture coupling
Writer: Hyeim Jeong
In SPR experiments, immobilization methods can be largely divided into two types.
Amine coupling uses the amine group present in the protein to covalently immobilize the sensor chip surface. On the other hand, the capture method immobilizes the protein using the affinity between the tag and a specific molecule.
The appropriate immobilization method is determined by the characteristics of the ligand or binding. Amine coupling is the most common and should be considered first. However, since it is not a oriented immobilization method, the ligand with the exposed binding site can fall by up to 10% compared to the entire immobilized ligand. On the other hand, the capture method using a tag performs oriented protein immobilization. If the binding site is far from the tag, the binding site can be efficiently exposed. However, since it is a non-covalent binding method, it has the disadvantage of lower surface stability than amine coupling. We introduce the capture coupling method that can complement the shortcomings of each immobilization method.
Capture coupling immobilization
Capture coupling is an immobilization method that combines his-tag capture and amine coupling, and can be used for ligands with his-tags. On the NTA sensor chip, the ligand forms a non-covalent bond with ni-NTA in the direction of the his-tag, and then covalently binds to the amine group on the NTA activated with EDC/NHS. After that, ethanolamine-HCl is used to block the remaining active NTA. The ligand bound by non-covalent bonds is removed using EDTA.
Increased exposure of ligand binding sites and surface stability
Figure 1. Single cycle kinetic evaluation
Table 1. Baseline drift
Based on the sensorgram and table, the binding tendencies of the capture coupling and His-tag capture methods show no significant difference. However, the sensor surface binding affinity of ligand in capture coupling is significantly enhanced compared to his-tag capture.
As shown in Figure 1, analyte binding is observed in his-tag capture (red) and capture coupling (blue) where the ligand is properly oriented. These two immobilization methods were also evaluated to have kinetic values that is not much different in the kinetic evaluation. In contrast, analyte binding is not observed in amine coupling (green) because the ligand was immobilized in a random orientation. Therefore, capture coupling can be determined to immobilize the ligand in the his-tag orientation like his-tag capture.
Table 1 shows that capture coupling reduces baseline drift by approximately 5.2 times compared to his-tag capture, indicating higher surface stability. This is because the amine coupling forms a covalent bond to prevent the removal of the ligand. Since the surface is stable, there is no need to replenish the ligand for each analyte cycle, and depending on the ligand, a strong regeneration solution can also be used.
Finding a suitable immobilization method in SPR analysis is an essential process for obtaining good data. If you want to obtain a strongly bound ligand surface while immobilizing using a his-tag, it may be advantageous to use capture coupling. However, you should be careful that the acetate buffer used in amine coupling is acidic, which may damage the ligand protein.
*For further inquiries about the experimental methods introduced on this page, please contact sales@icluebio.co.kr.
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