Rao L, Jones D P, Nguyen L H, McMahan S A, Burgess R R
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison 53706, USA.
Anal Biochem. 1996 Oct 15;241(2):173-9. doi: 10.1006/abio.1996.0395.
We present a simple, rapid, and inexpensive method for mapping epitopes of monoclonal antibodies using His-tagged protein fragments. In essence, four steps are involved: (i) purify overproduced His-tagged protein from inclusion body; (ii) fragment the protein by partial chemical or enzymatic hydrolysis and isolate the His-tagged peptides with a Ni(2+)-chelate column to generate a "ladder" containing a distribution of peptide fragment sizes; (iii) fractionate the isolated peptides by SDS-polyacrylamide gel electrophoresis and probe the ladder by immunoblot analysis; (iv) determine the size of the fragments that do and do not bind the monoclonal antibody using size markers specific to the His-tagged protein being studied. We have applied this method successfully to map the epitope positions of known and new monoclonal antibodies to Escherichia coli sigma 70. We believe this method will find broad application.
我们提出了一种使用His标签蛋白片段绘制单克隆抗体表位的简单、快速且廉价的方法。本质上，该方法涉及四个步骤：（i）从包涵体中纯化过量表达的His标签蛋白；（ii）通过部分化学或酶促水解使蛋白片段化，并用镍离子螯合柱分离His标签肽，以生成包含不同大小肽片段分布的“阶梯”；（iii）通过SDS-聚丙烯酰胺凝胶电泳对分离出的肽进行分级分离，并通过免疫印迹分析探测该“阶梯”；（iv）使用针对所研究的His标签蛋白的大小标记物，确定与单克隆抗体结合和不结合的片段的大小。我们已成功应用此方法来绘制针对大肠杆菌σ70的已知和新单克隆抗体的表位位置。我们相信该方法将得到广泛应用。
