Assay Method Information

Assay Name:  Fluorescence Polarization Assay
Description:  For the fluorescence polarization experiments, 5 nM fluorescently labeled Bag-1L (Bag-1L(1-20), fluorescein isothiocyanate (FITC)-MAQRGGARRPRGDRERLGSR; Bag-1L(61-80), FITC-RGAAAGARRPRMKKKTRRRS) or SRC-2 peptide (FITC-HDSKGQTKLLQLLTTKSDQM) was incubated with serially diluted AR-LBD (4 to 0.4 μM) in binding buffer (50 mM NaPO4, pH 6.5, 50 mM KCl, 1 mM DTT) and 10 μM DHT. The samples were then equilibrated for 45 min at ambient temperature. For the competition experiments, 400 to 0.4 nM Bag-1L core (GARRPR) or SRC-2 peptides (HDSKGQTKLLQLLTTKSDQM) were incubated with 4 μM AR-LBD and 5 nM fluorescent Bag-1 or SRC-2 peptide. Binding was measured using polarization (excitation λ = 485 nm, emission λ = 530 nm) on a Synergy H1 hybrid reader (Biotek).
Affinity data for this assay

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