Competitive binding assay (IC50)

The IC₅₀ (concentration of peptide required to inhibit 50% of radioligand binding) was determined for αMSH-PEG-Cy5-C′ dots and Scr-MSH-PEG-Cy5-C′ dots in competitive binding assays with ¹²⁵I-(Tyr²)-NDP, a radioiodinated αMSH analog with picomolar affinity for the MC1-R receptor.⁵⁵ Murine melanoma B16F10 cells were plated at a density of 0.2 million/well in 24-well tissue culture plates and incubated overnight. Individual wells were incubated at 25°C for 3 h with approximately 50,000 cpm (62,480 dpm) ¹²⁵I-(Tyr²)-NDP in 0.3 mL binding medium (DMEM with 25 mM N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid) (HEPES), pH 7.4, 0.02% BSA, and 0.3 mM 1,10-phenanthroline) with concentrations of the αMSH-PEG-Cy5-C′ dots and Scr-MSH-PEG-Cy5-C′ dots ranging from 10⁻¹³ to 10⁻⁵ mol/L. The binding media was aspirated after incubation. Cells were rinsed with 0.5 mL of ice-cold pH 7.1, 0.02% BSA/0.01 M PBS twice and lysed with 0.5 mL of 1 M NaOH for 10 min. The radioactivity in the cells was collected and measured in a γ-counter. The IC₅₀ values of αMSH-PEG-Cy5-C′ dots and Scr-MSH-PEG-Cy5-C′ dots were calculated with GraphPad Prism version 4.0.