NTA-mediated protein capturing strategy in screening experiments for small organic molecules by surface plasmon resonance.
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.
Yoshitani
Naoei
N
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.
Saito
Kazuki
K
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.
Saikawa
Wakana
W
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.
Asanuma
Miwako
M
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.
Yokoyama
Shigeyuki
S
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.|Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo, Japan.|RIKEN Harima Institute at SPring-8, Hyogo, Japan.
Hirota
Hiroshi
H
Protein Research Group, RIKEN Genomic Sciences Center, Yokohama, Japan.
D001665:Binding Sites; D015374:Biosensing Techniques; D006639:Histidine / Q000737:chemistry; D006801:Humans; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D018909:src Homology Domains / Q000502:physiology*
D010455:Peptides; D006639:Histidine; D019869:Phosphatidylinositol 3-Kinases; D009571:Nitrilotriacetic Acid