Bromophenol blue

For protein isolation from whole larvae, they were homogenized in 100μl of 1% Triton-X 100 in 1X PBS buffer (pH7.2) supplemented with 1% mammalian Protein Inhibitor Cocktail (Sigma Aldrich, Cat# P8340) on ice. The homogenate was centrifuged at 16000 x g for 15 minutes at 4°C. The supernatant was mixed with equal volume of 2X Lamilli Buffer (100 mM TrisHCl (pH6.8); 4% SDS: Sigma Aldrich, Cat# L3771; 0.2% Bromophenol Blue, HIMEDIA, Cat# MB123; 20% Glycerol and 200 mM β-mercaptoethanol, Sigma Aldrich, Cat# M3148), boiled at 95°C for 5 minutes and stored at -20°C. The protein quantitation was done by Bradford Assay (Biorad, Cat# 5000006). 1 μl of protein sample was incubated in 1 ml of five times diluted Bradford reagent, incubated for 5 minutes at RT and the OD of the mix was measured at 595 nm in spectrophotometer (Eppendorf, BioPhotometer Plus). The concentration of protein was calculated using a standard curve prepared freshly. For protein isolation from larval fat bodies, fat bodies were isolated from about 20 larvae directly into 1:1 mix of 2X Lamilli Buffer and 1X PBS supplemented with 1% mammalian Protein Inhibitor Cocktail (Sigma Aldrich, Cat# P8340) on ice. The rest of the steps followed were identical to that mentioned above.