Measuring β-galactosidase Activity in Drosophila

Embryos were collected overnight (16 hours), dechorionated in 3% hypochlorite solution for 2 minutes, washed thoroughly with water, and frozen in liquid nitrogen. 20 wandering third instar larvae and six adult flies (3 days post eclosion, 3 males and 3 females), respectively, were collected per replicate and frozen in liquid nitrogen. Frozen samples were homogenized on ice in assay buffer (1 mM MgSO₄, 2% Triton X-100 (v/v), 100 mM Hepes pH 8) supplemented with cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche). The pH 8 of assay buffer and Hepes were used to select for LacZ protein activity compared to endogenous β-galactosidase^{52 (link), 53 (link)}. Homogenates were spun twice at 15′000 rpm for 10 minutes at 4 °C, each time discarding debris and retaining supernatants. Homogenate protein concentrations were measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 1 µg of protein from homogenates was incubated at 37 °C for 50 minutes in 0.1 ml of assay buffer supplemented with 4-MUG (Sigma, M1633) at 0.9 mM final concentration. ß-galactosidase converts non-fluorescent substrate 4-MUG into 4-MU, a fluorescent product. 4-MU fluorescence was measured using EnVision Multilabel Reader 2104 (PerkinElmer).