Wedege E, Svenneby G
J Immunol Methods. 1986 Apr 17;88(2):233-7. doi: 10.1016/0022-1759(86)90010-4.
The effects of the blocking agents bovine serum albumin and Tween 20 in buffers at pH values 7.2 and 10.2 were compared in immunoblotting with 2 different antisera. The antisera were raised against a purified brain-specific protein fraction from human brain, soluble in perchloric acid, and phosphate-activated glutaminase from pig brain, respectively. The antigens were a crude perchloric acid-soluble brain extract, a crude brain phosphate-activated glutaminase fraction, and proteins commonly used as molecular weight markers. The binding patterns of the 2 antisera to the respective brain antigen preparations changed, depending on the blocking agent and the pH of the blocking buffer. Also, antibody binding to the molecular weight marker proteins was observed with some of the blocking buffers. Immunoblotting with Tris-saline, pH 10.2, containing 3% bovine serum albumin as blocking agent and diluting buffer for the antisera, showed negligible antibody binding to the marker proteins and most specific binding to the brain antigens.
在免疫印迹实验中，使用两种不同的抗血清，比较了封闭剂牛血清白蛋白和吐温20在pH值为7.2和10.2的缓冲液中的效果。这两种抗血清分别是针对从人脑中纯化得到的、可溶于高氯酸的脑特异性蛋白组分，以及猪脑的磷酸激活型谷氨酰胺酶产生的。抗原包括粗制的可溶于高氯酸的脑提取物、粗制的脑磷酸激活型谷氨酰胺酶组分，以及常用作分子量标准物的蛋白质。两种抗血清与各自脑抗原制剂的结合模式有所变化，这取决于封闭剂和封闭缓冲液的pH值。此外，在某些封闭缓冲液中还观察到抗体与分子量标准物蛋白的结合。用含有3%牛血清白蛋白作为封闭剂且用作抗血清稀释缓冲液的pH 10.2的Tris-盐水进行免疫印迹时，显示抗体与标准物蛋白的结合可忽略不计，且与脑抗原的结合最为特异。
