Bourassa C, Chapdelaine A, Roberts K D, Chevalier S
Department of Medicine, University of Montréal, Québec, Canada.
Anal Biochem. 1988 Mar;169(2):356-62. doi: 10.1016/0003-2697(88)90295-3.
Following their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, labeled proteins obtained from cultured canine prostatic epithelial cells incubated with [35S]-methionine and [32P]phosphate were subjected to alkali treatment, a method that is currently used to detect phosphotyrosine-containing proteins. Significant amounts of 35S-labeled material were lost during the alkali treatment. The crosslinking of proteins within the gels by glutaraldehyde treatment eliminated protein losses and did not alter the efficiency of phosphoester bond hydrolysis by alkali treatment. Consequently, the time required to detect alkali-resistant phosphoproteins by autoradiography was greatly reduced. Prostatic phosphoproteins were also shown to contain phosphotyrosine, indicating the presence of tyrosine protein kinase activity in these proliferating epithelial cells.
在用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离后，将用[35S] -甲硫氨酸和[32P]磷酸盐孵育的培养犬前列腺上皮细胞获得的标记蛋白质进行碱处理，这是目前用于检测含磷酸酪氨酸蛋白质的方法。在碱处理过程中，大量的35S标记物质丢失。通过戊二醛处理使凝胶内的蛋白质交联，消除了蛋白质损失，并且没有改变碱处理对磷酸酯键水解的效率。因此，通过放射自显影检测耐碱磷蛋白所需的时间大大缩短。前列腺磷蛋白也被证明含有磷酸酪氨酸，表明这些增殖的上皮细胞中存在酪氨酸蛋白激酶活性。
