ISOLATION OF PROTEINS FOR PEPTIDE-MAPPING, AMINO-ACID ANALYSES, AND SEQUENCING USING DISULFIDE CROSSLINKED POLYACRYLAMIDE GELS
ISOLATION OF PROTEINS FOR PEPTIDE-MAPPING, AMINO-ACID ANALYSES, AND SEQUENCING USING DISULFIDE CROSSLINKED POLYACRYLAMIDE GELS
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DOI：
10.1016/0003-2697(88)90497-6
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发表时间：
1988-06-01
影响因子：
2.9
通讯作者：
OLSON, MOJ
中科院分区：
文献类型：
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作者：
GHAFFARI, SH;DUMBAR, TS;OLSON, MOJ
已经检查了从二硫化物交联的聚丙烯酰胺凝胶中回收少量蛋白质（1-50 μ g）的条件。程序开发的溶解和沉淀的考马斯亮蓝染色的蛋白质带切离凝胶电泳分离后。然后将沉淀的蛋白重新溶解用于肽图谱、氨基酸分析或微测序。标准蛋白质（牛白蛋白、卵清蛋白、磷酸化酶B和β-D）的氨基酸组成半乳糖苷酶）与相应的常规纯化蛋白的值非常一致。在100 pmol或以下的样品上以高重复产率进行测序。该方法已成功应用于几种蛋白质和蛋白质片段。
Conditions for recovery of small amounts of proteins (1-50 .mu.g) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and .beta.-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.