A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.

When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM.

Application of green fluorescent protein to affinity electrophoresis; affinity of IgG-binding domain C from streptococcal protein G to mouse IgG1.

Affinity electrophoresis (AEP) using green fluorescent protein (GFP) was studied. We constructed a fusion protein that linked S147PGFP and IgG binding domain C from streptococcal protein G (GFP-SpGC). The affinity of GFP-SpGC for mouse IgG1 was measured.