看到一篇文献里蛋白提取方法，完全被折服了。太强悍了，蛋白还可以这样提。我以前一直以为裂解液裂解后，沉淀里面没有蛋白了，现在才发现沉淀里还含有大量不溶于裂解液里去污剂的蛋白。我以前是裂解细胞，离心，测浓度，然后直接往整管里加loading buffer，再离心取上清，看过这个步骤之后才知道，加loading buffer后沉淀里的许多蛋白会再次溶解。同一种蛋白可能存在溶于不同去污剂的成份。最后我有一个问题，一瓶细胞按下列步骤提取的蛋白后，能否比较该瓶中同一蛋白在含不同去污剂的裂解液中含量的多少？怎样选择内参？
For cell fractionation analysis, a general protocol was used which allowed for separation of cells into cytoplasmatic, nuclear and membrane/cytoskeleton fractions. Briefly, cells were harvested with a rubber policeman in chilled 50 mM Hepes, 150 mM NaCl, 3 mM MgCl2, measured pH 7.5, plus inhibitors, and ruptured by repeated passage through a 21-gauge needle. Nuclei were collected by centrifugation at 1000 ×g for 3 min and lysed with 1% SDS-lysis buffer. Centrifugation of supernatants at 21,460 ×g for 1 h at 4 °C gave membrane/cytoskeleton as pellet and the cytosolic fraction as supernatant. Membrane/cytoskeleton pellets were then resuspended as whole, SDS-solubilized membrane/cytoskeleton fraction (Tot) with 1% SDS-lysis buffer. Otherwise, membranes were fractionated into Tx-soluble (Sol) and Tx-insoluble (Insol) phases by treatment with TritonX-100-buffer (100 mM phosphate buffer, 150 mM NaCl, measured pH 8, 1.5% TritonX-100, 10% glycerol, plus inhibitors). Centrifugation at 21,460 ×g for 60 min gave a supernatant constituted by the Tx-soluble membrane phase and a residue pellet representing the Tx-insoluble, cytoskeleton-linked membrane phase which was resuspended in SDS-buffer.
