Conjugation of chelating agents to proteins and radiolabeling with trivalent metallic isotopes
Conjugation of chelating agents to proteins and radiolabeling with trivalent metallic isotopes
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DOI：
10.1038/nprot.2006.49
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Mather, Stephen J.
中科院分区：
文献类型：
--
作者：
Cooper, Maggie S.;Sabbah, Emmanuelle;Mather, Stephen J.
肽和蛋白质可以用金属元素标记，以便将它们用作成像报告物或用于其他应用。感兴趣的多肽首先与合适的螯合剂缀合，该螯合剂与感兴趣的元素形成稳定的复合物。该缀合步骤在水性或非水性条件下进行，这取决于底物的溶解度。对于大小大于类似于10 kDa的多肽，这通常在水性介质中进行。最常见的螯合剂与赖氨酸氨基反应。首先将蛋白质脱盐到pH 8 - 9的合适缓冲液中，并加入摩尔过量的双官能螯合剂。在适当的温育时间后，除去过量的、未反应的或水解的螯合剂，并将蛋白质缀合物脱盐到酸性缓冲液中。然后可以通过添加合适的金属盐，然后如果需要，通过去除未螯合的金属来标记缀合物。如以下方案中所述，整个缀合、纯化和标记过程需要约2天。
Peptides and proteins may be tagged with metallic elements in order to use them as imaging reporters or for other applications. The polypeptide of interest is first conjugated to a suitable chelating agent that forms stable complexes with the element of interest. This conjugation step is undertaken either in aqueous or in non- aqueous conditions depending on the solubility of the substrate. For polypeptides of greater than similar to 10 kDa in size, this is normally done in aqueous medium. Most commonly the chelators are reacted with lysine amino groups. The protein is first desalted into a suitable buffer at pH 8 - 9 and a molar excess of a bifunctional chelating agent is added. After a suitable period of incubation, excess, unreacted or hydrolyzed chelator is removed and the protein conjugate is desalted into an acidic buffer. The conjugate can then be tagged by addition of a suitable metal salt followed, if necessary, by removal of unchelated metal. As described in the protocol that follows, the entire conjugation, purification and labeling procedure takes about 2 d.