Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel.
Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel.
复制标题
在聚凝胺涂层的玻璃纤维片上进行蛋白质印迹。
DOI：
--
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
M. V. Montagu
中科院分区：
文献类型：
--
作者：
Joël Vandekerchove;G. Bauw;M. Puype;Jozef Van Damme;M. V. Montagu
已经开发了一种程序，它允许固定在玻璃纤维板上涂有聚季胺，聚凝胺，蛋白质和蛋白质片段先前分离的含十二烷基硫酸钠的聚丙烯酰胺凝胶。转移基本上如用于硝酸纤维素膜上的蛋白质印迹那样进行[Towbin，H.，Staehelin，T.和Gordon，J.（1979）Proc. Natl Acad. Sci. USA 76，4350-4354]，但现在用于确定固定化蛋白质的氨基酸组成和部分序列。蛋白质转移可以在用考马斯蓝染色凝胶中的蛋白质之后进行，通过考马斯蓝，固定的蛋白质作为蓝点可见，或者在没有预先染色的情况下进行，之后转移的蛋白质在与荧光胺反应后作为荧光点检测。发现后一种方法更有效，产生+/-20微克/cm 2的结合容量。丁胺检测的灵敏度等于或高于凝胶中蛋白质的经典考马斯染色。固定化的蛋白质可以水解时，仍然存在于玻璃纤维和可靠的氨基酸组合物，获得各种参考蛋白质固定在小于100 pmol的量。此外，更重要的是，玻璃纤维结合的蛋白质可以通过简单地切掉携带固定化蛋白质的片材区域并将盘安装在气相测序仪的反应室中而经受Edman降解程序。该固定化测序技术的结果显示为固定化的肌红蛋白（1 nmol）和两个肌动蛋白的蛋白水解片段（各+/-80 pmol），这些片段先前在含十二烷基硫酸钠的凝胶上分离。
A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.
影响因子：
2.9
作者：
DRAGER, RR;REGNIER, FE
通讯作者：
REGNIER, FE
使用酶联胶原蛋白结合纤连蛋白片段进行电泳后胶原蛋白的特异性检测。
影响因子：
2.9
作者：
Bell,ML;Engvall,E
通讯作者：
Engvall,E