Amphipod protein content under simulated invasion stress in wetlands

Every two weeks during the 6-week acclimation period, 8 adults were randomly selected from each tank for protein analysis (n = 8). Individuals were sacrificed in dry ice and stored at -80 °C. Whole frozen amphipods were prepared for protein quantification using a colorimetric assay (Thermo Scientific Pierce BCA Protein Assay Kit). Samples were homogenized with a Pro200 Bio-Gen Series homogenizer for 10 seconds on ice with the homogenization buffer (100 mmol l⁻¹ Tris-HCl, pH 7.5; 0.1% SDS (w/v), 0.5 mol l⁻¹ EDTA) containing a combination of protease inhibitors: 0.7 μg ml⁻¹ pepstatin A, 0.5 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ aprotinin and 20 μg ml⁻¹ phenylmethylsulfonyl fluoride at a dilution ratio of 1:20. In several instances, protein concentration at a 1:20 dilution was too high for the range of measurement: seven individuals were further diluted to 1:30 and two individuals were further diluted to 1:35. Homogenates were centrifuged at 13,000 g for 10 minutes. 50 μl of the resulting supernatant was transferred into a new 0.5 ml tube to use for total protein quantification. 10 μl of each sample was added to triplicate wells in 96 well plates. 200 μl of the BCA reagent was added to the wells and then placed on the shaker for 30 seconds. Plates were heated at 37 °C for 30 minutes and placed on the spectrometer at 562 nm. Protein values were standardized with 2 mg ml⁻¹ BSA standard.