Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.
Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.
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通过将小的、确定的底物与凝胶基质化学连接，扩大酶谱法的用途。
DOI：
10.1101/gr.1277303
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发表时间：
2003
期刊：
影响因子：
7
通讯作者：
K. McDowall
中科院分区：
文献类型：
--
作者：
V. Kaberdin;K. McDowall
在后基因组时代，对代谢和信号通路的全面蛋白质组学分析不可避免地面临大规模鉴定和表征具有特定酶活性的多肽的挑战。以前的工作已经表明，使用凝胶内活性染色(酶图谱)可以将微生物、植物和动物来源的各种酶活性分配给单个多肽。然而，一些限制，如特殊的底物要求，缺乏标准程序，以及难以区分具有重叠活性的酶，阻碍了酶谱作为常规实验室方法的广泛使用。在这里，我们证明，通过使用共价连接到凝胶基质上的小定义底物，我们可以在很大程度上克服上述问题，并在标准的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后很容易地测定凝胶中的许多不同类别的酶活性。此外，这一发展与蛋白质的二维分离兼容，因此在高通量筛选和表征复杂的生物和临床样本方面具有巨大的潜力。
In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.
用于微生物蛋白酶检测和表征的酶谱技术。
影响因子：
--
作者：
Lantz,MS;Ciborowski,P
通讯作者：
Ciborowski,P
蛋白质组的质谱分析。
DOI：
--
发表时间：
2001
期刊：
Molecular pharmacology.
影响因子：
--
作者：
Bakhtiar,R;Nelson,RW
通讯作者：
Nelson,RW