Effect of storage temperature and protease inhibitor on ß-galactosidase extract enzyme activity and protein concentration
Description
Extraction buffer omitting protease inhibitor was prepared and 1 protease inhibitor tablet was dissolved in 20ml. The extracts were prepared as follows: six flies expressing lacZ (DJ694,Bg2 recombinant strain BDSC:8176, BDSC:1776) were each ground in 50µl of buffer without protease inhibitor. The extracts were centrifuged and 40µl of supernatant was recovered from each fly and pooled (240µl total). The extract was split into two tubes (120µl each) and 120µl of +protease inhibitor buffer was added to one tube and 120µl of -protease inhibitor buffer was added to the other tube. Each extract condition was measured at 0h. From each extract tube 60µl was removed and stored on ice, 60µl was removed and stored at 4℃, and 5 aliquots of 12µl each were removed and stored at -20℃. The remaining extract from each tube was stored at room temperature. Measurements from each temperature condition were taken at 6, 24, 48, 72h. The extract preparations and measurements were done in triplicate. Independent extract preparations were made to measure enzyme activity (CPRG assay) and protein content (Bradford assay). Replicates 1,2,3 were used for the ß-gal activity measurements and Replicates 4,5,6 were used for the Protein content measurements. Extracts stored at 25°C were left on the bench, 0°C were stored on ice, 4°C were stored in the fridge, -20°C were stored in the freezer. Raw microplate readings are available upon request.