使用说明：
1. 准备溶液：室温融解试剂盒中的各种溶液，溶解后立即置于冰上并混匀。第一次使用时，把1.5ml
    PMSF(溶剂)加入到PMSF(晶体)中，溶解并混匀，即得到1.5ml 100mM PMSF。配制好的100mM PMSF溶
    液-20℃保存。如果最终目的是制备线粒体蛋白样品，根据样品数量，取适量线粒体分离试剂备用，
    在线粒体分离试剂加入到细胞样品中前数分钟内加入PMSF，使PMSF的最终浓度为1mM。取适量线粒体
    裂解液备用，在线粒体裂解液加入到线粒体样品中前数分钟内加入PMSF，使PMSF的最终浓度为1mM。
2. 收集细胞：
    对于贴壁细胞：用PBS洗一遍，用胰酶细胞消化液(Trypsin-EDTA Solution)消化细胞，离心收集细
    胞。胰酶细胞消化液(C0201)可以向碧云天订购。
    对于悬浮细胞：直接离心收集细胞。
3. 洗涤细胞：用冰浴预冷的PBS轻轻重悬细胞沉淀，取少量细胞用于计数，剩余细胞600g，4℃离心5
    分钟沉淀细胞。弃上清。
4. 预处理：加入1-2.5ml线粒体分离试剂或临用前添加了PMSF的线粒体分离试剂至2000-5000万细胞
    中，轻轻悬浮细胞，冰浴放置10-15分钟。
5. 匀浆：把细胞悬液转移到一适当大小的玻璃匀浆器中，匀浆10-30下左右。
6. 匀浆效果的鉴定：不同细胞不同匀浆器所需的匀浆次数有所不同，需自行优化。通常可以在匀浆10
    次后取约2微升细胞匀浆，加入30-50微升台盼蓝染色液，混匀后显微镜下观察台盼蓝染色阳性(蓝
    色)细胞的比例。如果阳性细胞比例不足50%，增加5次匀浆。随后再同前取样进行台盼蓝染色鉴定。
    当阳性比例超过50%时即可停止匀浆进入下一步。请勿过度匀浆，过度匀浆会导致线粒体的机械损
    伤。同时记录对于该细胞的匀浆次数，通常在后续实验时不必再摸索匀浆次数。
7. 把细胞匀浆在600g，4℃离心10分钟。
    注：如需获得纯度更高的线粒体，可以将此步骤的离心速度改为1000g，其它离心条件不变；获得
    更高纯度线粒体的缺点是相同数量细胞的线粒体抽提得率会下降。
8. 小心把上清转移到另一离心管中，在11,000g，4℃离心10分钟。
    注：如需获得纯度更高的线粒体，可以将此步骤的离心速度改为3500g，其它离心条件不变；获得
    更高纯度线粒体的缺点是相同数量细胞的线粒体抽提得率会下降。
9. 小心去除上清。沉淀即为分离得到的细胞线粒体。
    注：如果希望获得去除线粒体的细胞浆蛋白，在本步骤需收集上清，并且在收集上清时注意勿触及
    沉淀。随后把收集的上清12000g，4℃离心10分钟。取上清即为去除线粒体的细胞浆蛋白。
10. 线粒体的使用：
    10A. 如果用于完整线粒体的功能或酶活性研究，初始2000-5000万细胞分离得到的线粒体样品中可
         以加入150-200微升线粒体储存液，重悬线粒体。
    10B. 如果用于线粒体的蛋白分析，初始2000-5000万细胞分离得到的线粒体样品中可以加入150-200
         微升临用前添加了PMSF的线粒体裂解液裂解线粒体。裂解后的线粒体可以用于PAGE、
         Western、IP以及线粒体中的一些酶活性的测定等。裂解后的蛋白样品可以使用BCA法测定蛋白
         浓度。BCA蛋白浓度测定试剂盒(P0009/P0010/P0010S/P0011/P0012/P0012S)可以向碧云天订
         购。
    10C. 如果用于双向电泳，请使用适当的用于双向电泳的裂解液处理线粒体。

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61.Hao Y, Ren J, Liu C, Li H, Liu J, Yang Z, Li R, Su Y.
   Zinc Protects Human Kidney Cells from Depleted Uranium-induced Apoptosis.
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   Triptolide-induced oxidative stress involved with Nrf2 contribute to cardiomyocyte
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   Mimic of manganese superoxide dismutase induces apoptosis in human acute myeloid
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64.Yan J, Liu XH, Han MZ, Wang YM, Sun XL, Yu N, Li T, Su B, Chen ZY.
   Blockage of GSK3β-mediated Drp1 phosphorylation provides neuroprotection in neuronal
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   Neurobiol Aging. 2014 Aug 8. pii: S0197-4580(14)00518-1. doi: 10.1016/
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