Density Gradient Ultracentrifugation to Isolate Endogenous Protein Complexes after Affinity Capture.
Density Gradient Ultracentrifugation to Isolate Endogenous Protein Complexes after Affinity Capture.
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DOI：
10.1101/pdb.prot087957
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发表时间：
2016-07-01
影响因子：
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通讯作者：
Rout, Michael P
中科院分区：
文献类型：
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作者：
Fernandez-Martinez, Javier;LaCava, John;Rout, Michael P
本方案描述了通过密度梯度超速离心分离天然蛋白质复合物的方法。亲和捕获和天然洗脱实验的结果通常是以下物质的混合物：（1）在特定捕获条件下与目标蛋白质相关的复合物；（2）在纯化过程中因降解或拆解而产生的复合物片段；（3）用于天然洗脱样品的蛋白酶或试剂。为了分离这些成分并分离出均一的复合物，需要额外的纯化步骤。速率区带密度梯度超速离心是一种基于颗粒的流体动力学体积分离颗粒的可靠且强大的技术。密度梯度是通过混合合适的低分子量惰性溶质（例如蔗糖或甘油）的低密度和高密度溶液而产生的。该梯度是在一种溶剂中形成的，这种溶剂可以是用于亲和捕获和天然洗脱的任何溶剂，并且应该有助于保持复合物的结构和活性。
This protocol describes the isolation of native protein complexes by density gradient ultracentrifugation. The outcome of an affinity capture and native elution experiment is generally a mixture of (1) the complex(es) associated with the protein of interest under the specific conditions of capture, (2) fragments of the complex generated by degradation or disassembly during the purification procedure, and (3) the protease or reagent used to natively elute the sample. To separate these components and isolate a homogeneous complex, an additional step of purification is required. Rate-zonal density gradient ultracentrifugation is a reliable and powerful technique for separating particles based on their hydrodynamic volume. The density gradient is generated by mixing low- and high-density solutions of a suitable low-molecular-weight inert solute (e.g., sucrose or glycerol). The gradient is formed in a solvent that could be any of the solvents used for the affinity capture and native elution and should help to preserve the structure and activity of the assembly.