| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | SH2 domains were dialyzed into binding buffer (50 mM Hepes, pH 7.25, 150 mM NaCl, 0.01% Nonidet P-40, 5% glycerol) and a 30-μl volume serially diluted into pre-chilled 384-well low flange black flat bottom non-binding microplates (Corning) while on ice. 5 μl of 35 nM fluorescent peptide dissolved in binding buffer was added to each well (final concentration 5 nM), mixed 5 times by pipetting, and plates were incubated at 4 °C for 40 min. Fluorescence polarization experiments were performed with an Analyst AD (Molecular Devices, Sunnyvale, CA) spectrofluorimeter. Each well was excited using 485 nm light and emission was read at 530 nm. Fluorescence polarization was measured 1 mm from the bottom of each well with an integration time of 560 ms. All experiments were conducted in triplicate. |
| Affinity data for this assay | |
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