Kramberger-Kaplan Lukas, Austerlitz Tina, Bohlmann Holger
Department of Crop Sciences, Institute of Plant Protection, University of Natural Resources and Life Sciences, 1180 Vienna, Austria.
Methods Protoc. 2020 May 8;3(2):37. doi: 10.3390/mps3020037.
A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in , purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of and purified. GFP without tags was also expressed in and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different compartments for antibody production and affinity chromatography.
本文介绍了一种用于阳性选择针对作为融合蛋白表达的靶蛋白的特异性抗体以制备抗血清的方法。作为概念验证，融合蛋白FLAG::His::GFP::His::FLAG在[具体表达系统]中表达、纯化，并用于免疫兔子。通过蛋白A亲和法对获得的血清进行预清除。CusF::FLAG融合蛋白在[具体表达系统]的周质中表达并纯化。无标签的GFP也在[具体表达系统]中表达并通过有机萃取纯化。然后将这些蛋白偶联到NHS活化的琼脂糖上，用于阳性选择抗GFP和抗FLAG抗体。在蛋白质印迹中测试获得的血清对不同蛋白质样品和融合蛋白的特异性。通过单一亲和色谱步骤可实现抗体的高特异性。一般来说，我们建议为了抗体生产和亲和色谱，用不同的标签并在不同的[具体表达系统]区室中表达靶蛋白。
