NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance |
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Authors: | Yoshitani Naoei Saito Kazuki Saikawa Wakana Asanuma Miwako Yokoyama Shigeyuki Hirota Hiroshi |
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Affiliation: | Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan. |
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Abstract: | Nitrilotriacetate (NTA)-mediated capture of a histidine-tagged protein is widely used as an easy and simple method to reversibly immobilize the protein onto a sensor chip for surface plasmon resonance (SPR). However, in spite of its advantages, the NTA-capturing strategy is rarely employed for ligand screening experiments using SPR, because it was thought to cause substantial errors in binding responses, due to the inevitable protein dissociation during the monitoring period. In this study, as demonstrated in a ligand screening for the histidine-tagged SH3 domain of the human phosphatidylinositol 3-kinase p85alpha subunit, false responses after adhesion of undesirable compounds to a target protein could be minimized with the NTA strategy, while binding responses of a positive control peptide still stayed within a 1%-deviation against the theoretical binding capacity. |
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Keywords: | Histidine‐tagged protein Ligand screening Nitrilotriacetate SH3 domain Surface plasmon resonance |
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