Greenfield Edward A, DeCaprio James, Brahmandam Mohan
Cold Spring Harb Protoc. 2019 Dec 2;2019(12):2019/12/pdb.prot100008. doi: 10.1101/pdb.prot100008.
Some native proteins can be isolated in pure form from cell lysates or tissue preparation using SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Antigens purified this way often induce good antibody responses. There are many different ways to process a gel fragment containing the protein of interest for injection. Such samples can be processed into small pieces and then injected, either by fragmenting the gel by passing it repeatedly through a syringe, or drying the entire gel slice and grinding it into a powder. Injections using the whole gel fragment should only be used with larger animals such as rabbits. For mice or other small animals, electroelution or electrophoretic transfer of the protein should be used to prepare the protein for injection. In the latter method, the protein is transferred to a suitable membrane, such as nitrocellulose or PVDF. The location of the desired protein is identified by staining, and the protein band is then cut from the surrounding membrane, minced, and dissolved in a small amount of dimethyl sulfoxide before subcutaneous injection into the animal.
一些天然蛋白质可以通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）从细胞裂解物或组织制剂中以纯形式分离出来。以这种方式纯化的抗原通常能诱导良好的抗体反应。对于含有感兴趣蛋白质的凝胶片段，有许多不同的处理方法用于注射。这样的样品可以切成小块然后注射，要么通过反复将凝胶通过注射器使其破碎，要么将整个凝胶切片干燥并研磨成粉末。使用整个凝胶片段进行注射仅适用于较大的动物，如兔子。对于小鼠或其他小动物，应使用蛋白质的电洗脱或电泳转移来制备用于注射的蛋白质。在后一种方法中，蛋白质被转移到合适的膜上，如硝酸纤维素膜或聚偏二氟乙烯膜。通过染色确定所需蛋白质的位置，然后从周围的膜上切下蛋白质条带，切碎，并在皮下注射到动物体内之前溶解在少量二甲基亚砜中。
