Litovchick Larisa
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):2018/7/pdb.prot098418. doi: 10.1101/pdb.prot098418.
Most protein solutions can be mixed with sample buffer and analyzed directly by immunoblotting. Any protein extraction buffer or solvent that is compatible with gel electrophoresis can be used for preparing samples for immunoblotting. Examples of the protein solutions suitable for immunoblotting include purified proteins, detergent lysates of cells, samples prepared by subcellular fractionation, and protein eluates from chromatography columns. The protein of interest must be sufficiently abundant in the sample to allow for efficient detection, but the total amount of protein in the sample should not exceed the loading capacity of the gel. For this reason, protein antigens of very low abundance must be partially purified from the protein mixtures containing high concentrations of the contaminating proteins. In this protocol, solutions containing the protein antigens of interest are mixed with SDS-PAGE sample buffer and boiled to prepare samples for protein gel electrophoresis.
大多数蛋白质溶液可与样品缓冲液混合，直接通过免疫印迹法进行分析。任何与凝胶电泳兼容的蛋白质提取缓冲液或溶剂都可用于制备免疫印迹样品。适用于免疫印迹的蛋白质溶液示例包括纯化的蛋白质、细胞的去污剂裂解物、通过亚细胞分级分离制备的样品以及来自色谱柱的蛋白质洗脱液。目标蛋白质在样品中必须足够丰富，以便进行有效检测，但样品中的蛋白质总量不应超过凝胶的上样量。因此，必须从含有高浓度污染蛋白质的蛋白质混合物中部分纯化极低丰度的蛋白质抗原。在本实验方案中，将含有目标蛋白质抗原的溶液与SDS-PAGE样品缓冲液混合并煮沸，以制备用于蛋白质凝胶电泳的样品。
