COVALENT IMMOBILIZATION OF PROTEINS FOR HIGH-SENSITIVITY SEQUENCE-ANALYSIS - ELECTROBLOTTING ONTO CHEMICALLY ACTIVATED GLASS FROM SODIUM DODECYL-SULFATE POLYACRYLAMIDE GELS
COVALENT IMMOBILIZATION OF PROTEINS FOR HIGH-SENSITIVITY SEQUENCE-ANALYSIS - ELECTROBLOTTING ONTO CHEMICALLY ACTIVATED GLASS FROM SODIUM DODECYL-SULFATE POLYACRYLAMIDE GELS
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DOI：
10.1021/bi00418a031
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发表时间：
1988-09-06
期刊：
影响因子：
2.9
通讯作者：
KENT, SBH
中科院分区：
文献类型：
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作者：
AEBERSOLD, RH;PIPES, GD;KENT, SBH
我们报告了一种制备适合高灵敏度 N 末端氨基酸序列分析形式的蛋白质的新方法。将通过聚丙烯酰胺凝胶电泳分离的蛋白质电泳转移到通过引入苯基异硫氰酸酯官能团进行化学活化的玻璃纤维滤纸上。在电转移过程中蛋白质与基质共价偶联。通过荧光染色或放射自显影检测含有转移蛋白的条带，从玻璃纤维过滤器上切下，并直接加载到气相测序仪的盒中。蛋白质和玻璃纤维支持物之间相互作用的共价性质允许使用更有力的固相测序方案和替代测序试剂。这种高效分离和共价偶联方法为增强灵敏度的蛋白质序列分析提供了必要的第一步。该方法已成功应用于从 SDS 聚丙烯酰胺凝胶中分离多种蛋白质，并被证明与标准 Edman 试剂异硫氰酸苯酯和替代测序试剂（如 4-(N,N''-二甲氨基）偶氮苯-4-''-异硫氰酸酯 (DABITC)）兼容。
We report a new method for the preparation of proteins in a form suitable for high-sensitivity N-terminal amino acid sequence analysis. Proteins separated by polyacrylamide gel electrophoresis were electrophoretically transferred onto glass fiber filter paper chemically activated by the introduction of phenyl isothiocyante functional groups. The proteins became covalently coupled to the matrix during the electrotransfer process. Bands containing transferred proteins were detected by fluorescent staining or autoradiography, cut out from the glass fiber filter, and directly loaded into the cartridge of a gas-phase sequenator. The covalent nature of the interactions between protein and glass fiber support permitted the use of more vigorous solid-phase sequencing protocols and of alternative sequencing reagents. This high-efficiency isolation and covalent coupling method provides the essential first step toward enhanced-sensitivity protein sequence analysis. The method has been successfully applied to the isolation of a wide variety of proteins from SDS-polyacrylamide gels, and was shown to be compatible with both the standard Edman reagent phenyl isothiocyanate and alternative sequencing reagents such as 4-(N,N''-dimethylamino)azobenzene-4-''-isothiocyanate (DABITC).