10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.
Frozen cell-pellets were resuspended in 1,7 ml IP-buffer with 8,5 µl PIC and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.
1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).
10 µg of each sample were used for SDS-PAGE (Westernblot).
5 µl of protein (in ureabuffer) were used.
Measured protein concentrations:
- Dark: 2,10 µg/µl
- Red: 2,49 µg/µl
- Green: 2,80 µg/µl
- Blue: 3,23 µg/µl