Solid-phase binding assays of peptides using EGFP-Src SH2 domain fusion protein and biotinylated Src SH2 domain.
Two solid-phase binding assays were designed and evaluated for their potential use in comparing the affinity of peptides to the Src SH2 domain. Resin beads attached to peptides were incubated with the enhanced green fluorescence protein(EGFP)-Src SH2 domain fusion protein or the biotinylated Src SH2 domain and extensively washed. The beads-attached tetrapeptides with high affinities to the EGFP-Src SH2 domain showed more fluorescence intensity than those beads containing tetrapeptides with weak binding affinities, as shown by fluorescence microscopy and fluorescence imaging system. Only the beads attached to pYEEI produced a dark purple color on incubation of the beads, respectively, with the biotinylated Src kinases SH2 domain, alkaline phosphatase-coupled streptavidin, and BCIP/NBT. These solid-phase binding assays may have potential applications for the screening of peptides for the Src kinases SH2 domains.
Ye
Guofeng
G
Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA.
Ayrapetov
Marina
M
Nam
Nguyen-Hai
NH
Sun
Gongqin
G
Parang
Keykavous
K
D019921:Biotinylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019061:src-Family Kinases