8 eggs per day, day 7- day 13
cut CAM, wash in precooled PBS,
in 10 ml WASH 1 (PBS, 5 mM EDTA, COMPLETE) on ice
cut in pieces in petri dish on ice
centrifuge 4 min at 1000 rpm (table top centifuge, Falcon 15 ml tubes) at 4°C (precool Heraeus centrifuge in E2)
aspirate supernatant, add 10 ml WASH 2 (PBS, 5 mM EDTA, 5 μg/ml Leupeptin+ 0.5 mM Pefabloc SC)
centrifuge 4 min at 1000 rpm (table top centifuge, falcon 15 ml tubes) at 4°C
aspirate supernatant, add 5 ml WASH 2
centrifuge 4 min at 1000 rpm (table top centifuge, falcon 15 ml tubes) at 4°C
aspirate supernatant, add 1-1.5 ml WASH 2, transfer to preweighed 2 ml Eppendorf tubes, optional rinse Falcon tube with ca. 0.5 ml WASH 2
centrifuge 5 min at 2000 rpm (Eppendorf centrifuge) at 4°C (precool Eppendorf centrifuge in E7?)
aspirate supernatant, weigh and store at -20°C
- COMPLETE
Boehringer 1 697 498 20 tablets 345 CHF
- Leupeptin
Sigma L-0649 25 mg 185,90 CHF
- Pefabloc SC (AEBSF, 4-(2-Aminoethyl)-benzenesulfonyl fluoride, hydrochloride)
Boehringer 1 429 868 100 mg 75 CHF