Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates.
Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates.
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DOI：
10.1016/j.ab.2009.03.026
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发表时间：
2009-06
影响因子：
2.9
通讯作者：
Emiko Kinoshita-Kikuta;E. Kinoshita;T. Koike
中科院分区：
文献类型：
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作者：
Emiko Kinoshita-Kikuta;E. Kinoshita;T. Koike
抗磷蛋白抗体的低特异性通常是免疫印迹分析中的问题。我们引入了一种简单的细胞裂解物预处理程序，以在免疫印迹中更特异地检测磷蛋白。细胞磷蛋白被优先捕获的Phos标签琼脂糖磷酸盐亲和珠在自制的旋转离心微管单元，和非磷酸化的蛋白质被排除在滤液中。磷蛋白结合珠悬浮在样品加载染料溶液进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，然后进行蛋白质印迹。我们证明了通过这种新技术可以提高A431细胞裂解物中磷酸化Shc和丝裂原活化蛋白激酶亚型的检测。
The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loading dye solution were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by Western blotting. We demonstrated improved detection of phosphorylated Shc and mitogen-activated protein kinase isoforms in A431 cell lysates by this new technique.