| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | Three fluorescein-labeled peptide probes, FITC-sulfopeptide, FITC-phosphopeptide, and FITC-tyrosinepeptide (FITC, fluorescein isothiocyanate), were used. Individual peptide probes were dissolved in assay buffer containing potassium phosphate (20 mM, pH 7.35), NaCl (100 mM), DTT (2 mM), and bovine gamma globulin (0.1%). Aliquots of the peptide probe solution were distributed to 96-well fluorescence plate to reach a final concentration of 10 nM. After the addition of indicated amounts of SH2 domain protein, the assay solutions were mixed and incubated at RT in the dark for 25 min. Fluorescence polarization experimentswere performed on a Synergy H1 plate reader (BioTek Instruments, Inc.) equipped with standard filter cube (λEx = 485 nm, BP = 20 nm; λEm = 528 nm, BP = 20 nm). A standard sample layout in a 96-well plate was designed so that a single plate contains all the samples for one SH2 domain variant. Triplicate samples at each protein concentration were arrayed in the plat |
| Affinity data for this assay | |
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