使用 DSS 或 BS3 对相互作用的蛋白质进行交联。缓冲液条件和试剂用量参考了已发表的实验方法，但每种蛋白质相互作用的具体用量应进行优化。
Protocol
1. Suspend cells at ~25×106 cells/ml in PBS (pH 8.0).
2. Wash cells three times with ice-cold PBS (pH 8.0) to remove amine-containing culture media and extracellular proteins from the cells.
3. For cell–surface interaction studies, add ligands to the cells and incubate for 1 h at 4°C.
4. Dissolve DSS or BS3 in dry DMSO at a concentration of 25-mM. Note: BS3 may be added directly to PBS buffer or dissolved as a stock solution in DMSO.
5. Add an aliquot of the DSS or BS3 solution to the reaction medium to obtain a final concentration of 0.5 to 5-mM. Note: Simons et al. (1999)^[10.1091/mbc.10.10.3187]^ successfully used a concentration of 0.5-mM BS3 with Madin–Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) to crosslink the protein interaction complexes on the cell surfaces.
6. Incubate the reaction mixture for 30 min at room temperature. To reduce active internalization of BS3 into cells, this incubation may be performed at 4°C.
7. Quench the reaction by adding an aliquot of 1-M Tris, pH 7.5, to give a final concentration of 10 to 20-mM.
8. Incubate the quenching reaction for 15 min at room temperature.
9. Lyse cells and analyze the protein interactions by electrophoresis, western blotting, and mass spectrometry.
参考
Source: Greg T. Hermanson，Bioconjugate Techniques 3rd Ed., 2013, Academic press, 978-0-12-382239-0
