QUANTITATIVE ZYMOGRAPHY - DETECTION OF PICOGRAM QUANTITIES OF GELATINASES
QUANTITATIVE ZYMOGRAPHY - DETECTION OF PICOGRAM QUANTITIES OF GELATINASES
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DOI：
10.1006/abio.1994.1186
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发表时间：
1994-05-01
影响因子：
2.9
通讯作者：
STETLERSTEVENSON, WG
中科院分区：
文献类型：
--
作者：
KLEINER, DE;STETLERSTEVENSON, WG
酶谱法是一种电泳技术，用于鉴定在非还原条件下在聚丙烯酰胺凝胶中分离的酶的蛋白水解活性。它已被广泛用于肿瘤和细胞培养条件培养基中蛋白酶的定性评价。使用市售的预制凝胶和现代图像分析系统，我们已经评估了酶谱作为一种定量技术。明胶酶谱中明胶被纯化的明胶酶A（一种基质金属蛋白酶）消化的程度与10- 20倍范围内装载的酶的量成正比。在过夜（18小时）消化期内，该测定的线性范围从10 pg酶扩展到120 pg酶。消化的初始速率与酶加载量成比例，并且改变孵育时间导致测定的线性范围发生变化。明胶酶A的活性和潜伏形式在该测定系统中显示出相同的消化程度。这些结果证明了酶谱法在明胶酶活性定量评估中的使用，并证明了其作为分析存在的明胶酶种类的定性技术的有用性。(C)1994年出版社出版。
Zymography is an electrophoretic technique used to identify proteolytic activity in enzymes separated in polyacrylamide gels under nonreducing conditions. It has been used extensively in the qualitative evaluation of proteases present in tumors and cell culture conditioned media. Using commercially available precast gels and a modern image analysis system, we have evaluated zymography as a quantitative technique. The degree of digestion of gelatin within the zymogram by purified gelatinase A, a matrix metalloprotease, is directly proportional to the amount of enzyme loaded over a 10- 20-fold range. With an overnight (18 h) digestion period, the linear range of this assay extended from 10 to 120 pg of enzyme. The initial rate of digestion is proportional to the enzyme loading and varying the incubation time results in a shift in the linear range of the assay. Active and latent forms of gelatinase A show the same degree of digestion in this assay system. These results justify the use of zymography in the quantitative assessment of gelatinase activity as well as demonstrate its usefulness as a qualitative technique for the analysis of gelatinase species present. (C) 1994 Academic Press, Inc.