| Assay Method Information | |
| | Folate Receptor Binding Assay |
| Description: | Table 15: Folate receptor binding was measured in black 96-well plates using a fluorescence polarization assay. A fluorescent probe consisting of a folate analog linked to Cy5 (FR-Cy) was custom synthesized. Test compounds were resuspended in DMSO and 3-fold serial dilutions were made from 300-0.015 μM (100× final concentrations). Binding reactions were conducted in 200 μl final volume in 20 mM Hepes (pH 7.5) 250 mM NaCl 0.0150 Tween-20 1% DMSO with 2.5 nM folate receptor beta (Acro Biosystems) and 2 nM probe. Fluorescence polarization was measured using λex=620 nm, λem=688 nm on an Envision plate reader (Perkin Elmer) after 2 hr incubation time. Dose responses were conducted in duplicate and normalized to the response with DMSO (high) and 1 μM folic acid (low) on each plate. IC50 values were determined by fitting to 4-parameter curves in GraphPad Prism. |
| Affinity data for this assay | |
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