Gruber M Y, Cheng K H, Lepock J R, Thompson J E
Anal Biochem. 1984 Apr;138(1):112-8. doi: 10.1016/0003-2697(84)90777-2.
Modifications to the two-phase polymer gradient procedure for isolating plasma membrane from mammalian cells have resulted in greatly increased yields of purified plasma membrane. First, the cells were not treated with a membrane stabilizer (ZnCl2) prior to homogenization. This reduced the severity of homogenization required for disruption and allowed a greater proportion of the surface membrane to form large, flattened sheets that are more easily purified than the smaller fragments formed during more severe homogenization. Second, three crude fractions obtained from the homogenate (600g, 2000g, and 12,000g pellets), rather than a single, low-speed pellet (600g) containing only large sheets of membrane, were subjected to gradient centrifugation to obtain plasma membrane. This modification allowed purification of small as well as large fragments of plasmalemma and greatly increased the yield of purified membrane. Mg+2-dependent, Na+-K+-stimulated ATPase, a marker enzyme for plasma membrane, was enriched in the purified fraction by congruent to 17-fold relative to homogenate on a specific activity basis, and the yield of isolated plasma membrane averaged 70%, and was occasionally as high as 90%.
对用于从哺乳动物细胞中分离质膜的两相聚合物梯度法进行改进后，已大大提高了纯化质膜的产量。首先，细胞在匀浆前未用膜稳定剂（ZnCl₂）处理。这降低了匀浆破坏所需的强度，并使更大比例的表面膜形成大的扁平片层，与在更剧烈匀浆过程中形成的较小片段相比，这些片层更易于纯化。其次，将从匀浆物中获得的三个粗分级分（600g、2000g和12,000g沉淀），而不是仅包含大片膜的单个低速沉淀（600g），进行梯度离心以获得质膜。这种改进使得质膜的小片段和大片段都能得到纯化，并大大提高了纯化膜的产量。Mg²⁺依赖性、Na⁺-K⁺刺激的ATP酶是质膜的标记酶，在纯化级分中相对于匀浆物，其比活性富集了约17倍，分离的质膜产量平均为70%，偶尔高达90%。
