Melanin Extraction from Drosophila Females

The D. melanogaster strains used in this study, WT- CantonS (BDSC 64349), ebonyS (BDSC 498), and yw (BDSC 1495) were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN). All D. melanogaster strains were maintained on our standard fly food [86 (link)] on a 12 h light:dark photoperiod at 25°C and 80% relative humidity. Adult flies (4-7 day old) were collected and sexed for further processing. Acid-resistant material was collected from groups of 25 homogenized female flies. Samples were homogenized in 500 μL diH₂O using an electric pellet pestle cordless motor (Kimble). After homogenization, 500 μL of 12M HCl was added to the homogenate (6M final concentration). Samples were digested for 24 hours on an Eppendorf ThermoMixer C shaker at 85°C with 700 RPM shaking. After digestion, samples were spun down for 30 minutes at room temperature at 15,000 RCF. Samples were washed three times, first with 1 mL 1X PBS, then 1 mL 10% PBS, and finally 1 mL diH₂O. Washed melanin samples were lyophilized, weighed, and combined for ssNMR analysis.