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如何纯化酵母微粒体(microsome)?

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A:有人做过酵母microsome的纯化么?有没有比较常用的protocol分享。

B:

Briefly, the cells were harvested by centrifugation (3,750 rpm at 4℃ for 10 mins) and the cell pellet was washed with 100 mL of TES buffer (50 mM Tris–HCl, pH, 7.5, 1 mM EDTA, 0.6 M sorbitol).

The cells were centrifuged as above, resuspended in 100 mL of TES-M (TES supplemented with 10 mM 2-mercaptoethanol), and allowed to incubate at room temperature for 10 min. The yeast cells were centrifuged again at 3,750 rpm for 10 min, and the pellet was resuspended in 2.5 mL of extraction buffer (1% bovine serum albumin, fraction V, 2 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, all dissolved in TES).

Zirconia/silica beads (0.5 mm in diameter, Biospec Products) were added until skimming the surface of the cell suspension. Cell walls were disrupted manually by hand shaking in a cold room for 10 min at 30-s intervals separated by 30-s intervals on ice.

Cell extracts were transferred to a 50-mL centrifuge tube, the Zirconia/silica beads were washed three times with 5 mL of extraction buffer, and the washes were pooled with the original cell extracts. Finally, microsomes were obtained by differential centrifugation at 10,000g for 10 min at 4°C to remove cellular debris followed by centrifugation at 100,000g for 70 min at 4°C. The microsomal pellets were resuspended in 1.5 mL of TEG-M buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 20% glycerol, and 1.5 mM 2-mercaptoethanol) and stored frozen at -80 °C.

A: 谢谢!可以定量吗?

B:这个是文献里面的,buffer有些复杂,我一般用pbs代替。总蛋白用考马斯亮蓝法定量,反应文献建议总蛋白量是12.5 mg/ml

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