[討論] akaline phosphatase activity assay
大家好!
小第的老闆想要探討osteogenic differentiation
最近打算購買Alkaline Phosphatase Assay Kit (Colorimetric),Abcam
但該組kit沒有提到關於cell sample前處理方法
目前參考一些protocol跟paper,想請問板上的各位神人,下面設計的前處理方式是否ok?
1.wash monolayer cell in 24 well(1*10^4/well) with PBS twice
2.add 300ul 1% triton X-100 in PBS (lysis buffer)
3.scrapping cell and incubation on ice for 1.5 hr
4.2000g, 15min, 4℃ centrifugation
5.測定supernatant protein concentration
6.依Kit說明書測定Alkaline Phosphatase含量
另外還有一些關於此實驗的疑問
有些paper中有看到加了lysis buffer(0.1~0.2% triton X-100),會冷凍解凍三次
如果我用1% triton X-100還需要冷凍解凍嗎?
另外關於sample normalize有點疑惑,請問是測定total protein濃度後,
以lysis buffer將sample調整到相同濃度並取等量進行酵素活性測定(類似WB的定量方式)?
還是無須調整sample直接取等量跑完酵素活性測定後,再和total protein濃度相除?
麻煩各位協助了!
謝謝!
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