??Cell Lysates - Cells must be lysed before assaying according to the following directions.

??1. ?Adherent cells should be detached with trypsin and then collected by centrifugation ?(suspension cells can be collected by centrifugation directly).

??2. ?Wash cells three times in cold PBS.

??3. ?Resuspend cells in PBS (1×) and the cells was subject to ultrasonication for 4 times (or Freeze cells at ≤-20oC. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)

??4. ?Centrifuge at 1500×g for 10 minutes at 2 - 8oC to remove cellular debris.

??細胞裂解液：

??1）貼壁細胞需要先用胰酶消化，離心收集細胞（懸浮細胞可直接離心收集）；

??2）將收集到的細胞用冷PBS洗3次；

??3）物理方法裂解細胞（可先超聲破碎細胞，再反復凍融）：

??i 超聲破碎：取適量PBS重懸細胞，用一定功率的超聲波處理細胞懸液，使細胞急劇震蕩破裂。

??ii 反復凍融：將待破碎的細胞在-20oC以下冰凍，室溫融解，反復3次，使細胞溶脹破碎。

??4）將標本于2-8oC 1500×g離心10分鐘，收集上清備用。