以下方案基于已发表的方法以及Thermo Fisher Scientific的重原子交联剂的具体说明。 对于新的应用，必须优化添加到样品中的交联剂的量，以获得关于相互作用的有用信息。
Protocol
1. Dissolve the protein(s) to be studied in 20-mM HEPES buffer, pH 7.5, at a concentration of 5 to 10 μM.
2. Dissolve together in one solution the desired heavy and light crosslinker analogs in dry DMSO at an equal concentration of up to 100-mM. Use either the pair BS2G-d4/BS2G-d0 or BS3G-d4/BS3G-d0, but do not mix the different sized crosslinkers together. The heavy and light analogs of the same type should always be dissolved at equivalent concentrations in DMSO to prepare the stock solution.
3. Add a quantity of the crosslinker solution to the protein solution to obtain at least a 10-fold molar excess of the crosslinkers over the concentration of the protein. Studies should be performed at several levels of crosslinker addition to determine the optimal conjugation conditions (e.g., 10-, 50-, 100-, and 200-fold excess).
4. Quench the reaction by the addition of NH4HCO3 to a final concentration of 20-mM. The optimal time course for the reaction should be determined by removing portions of the solution at different points, starting at about 5 to 10 min and extending out to 2 h in length.
5. Analyze the quenched reaction by SDS electrophoresis, western blotting, and mass spectrometer analysis.
参考
Source: Greg T. Hermanson，Bioconjugate Techniques 3rd Ed., 2013, Academic press, 978-0-12-382239-0
