KOSEN 한인과학기술자네트워크

kit 사용하지 않고 동위원소 처리한 후에 계산 방법에 대해서 알려주세요 PROTOCOL: 1. Wash cells 2x in DMEM/0.1%BSA at 37°C 2. Do serum-free stepdown for 4 hours in DMEM/BSA 3. Wash cells 2x KRH/0.1% BSA, 37°C 4. Do glucose-free stepdown in 0.45ml KRH/0.1% BSA, 37°C 5.Add cytochalasin B to final 10mM at time of stimulation for non-specific uptake Add insulin add whatever concentration for 20 minutes 6. Last 5 minutes of insulin stimulation add 50ml of 10x START 7.At the end of incubation wash cells in 12-well plates by dunking into large beaker of ice-cold PBS. Put plates in ice and wash 2x in ice-cold PBS 8. Solubilize cells in 0.5ml 0.05% SDS 9. Count 0.4ml in scintillation fluid and count 50ml of 10X START for specific activity calculations 10. Save remaining lysates for protein determination. Plates can be stored by wrapping in Parafilm and storing at 4°C 11. Calculate specific activity of glucose uptake by doing protein assays and subtracting the cytoB cpm values from all wells. Express as xmol/mg/min Example calculation: --(0.1mmol/L glucose)(0.0005L)(0.4ml/0.5ml)=0.00004mmol=0.04mmol 2-deoxyglucose counted --if 50ml of START=300,000cpm, then one should use 300,000 cpm (0.4/0.5)=240,000 (since only 4/5 of well is counted) --240,000 cpm/0.04 mmole=6 x 106 cpm/mmol ---Then, for example, 6000cpm (after subtracting cyto B value) /6 x 106 cpm/mmol=0.001mmol=1nmole of 2-deoxyglucose uptake --assuming [prot]/well=0.2 mg and time of uptake= 4minutes, 1then 1nmol/0.2mg/4min=1.25 nmol/mg/min 계산식이 있는데.. 이해가 잘 되지 않습니다.. 왜 그렇게 푸는지가 이해가 안되는데요.. 명쾌한 답변 부탁드립니다.