Cold Spring Harb Protoc. 2020 Jun 1;2020(6):098475. doi: 10.1101/pdb.prot098475.
Detection of the protein antigens in immunoblots prepared with immunoprecipitated protein antigens can be affected by the presence of high amounts of the immunoprecipitating antibodies. When it is not possible to use the immunoprecipitating antibodies and the primary antibodies raised in different species, this protocol provides a convenient and inexpensive alternative to achieve optimal detection of immunoprecipitated protein antigens. In this protocol, a nitrocellulose or polyvinylidene fluoride membrane containing immunoprecipitated protein samples is rinsed with ultrapure HO after the transfer of proteins and detection of the total proteins using Ponceau S dye (optional). Blocking solution is applied to the membrane, and the membrane is incubated and then rinsed off (optional) before addition of the primary antibody labeled with biotin. After washing, the membrane is incubated with enzyme- or fluorochrome-labeled avidin for detection.
免疫印迹中免疫沉淀蛋白抗原的蛋白抗原检测可能受到免疫沉淀抗体大量存在的影响。当无法使用免疫沉淀抗体和在不同物种中产生的初级抗体时，本方案提供了一种方便且廉价的替代方法，可实现免疫沉淀蛋白抗原的最佳检测。在该方案中，在蛋白质转移和使用丽春红 S 染料（可选）检测总蛋白质后，用超纯水（HO）冲洗含有免疫沉淀蛋白样品的硝酸纤维素或聚偏二氟乙烯膜。将封闭溶液施加到膜上，在加入用生物素标记的初级抗体之前，将膜孵育并冲洗掉（可选）。洗涤后，将膜与酶或荧光素标记的亲和素孵育以进行检测。
