Cowan D A, Daniel R M
J Biochem Biophys Methods. 1982 Apr;6(1):31-7. doi: 10.1016/0165-022x(82)90023-9.
A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.
酪蛋白琼脂平板测定法用于嗜温蛋白酶和嗜热蛋白酶的定量测定。由于许多蛋白酶具有热稳定性，因此可以在较高温度下进行测定。平板测定法的灵敏度随温度升高而增加，最佳测定温度取决于酶的热稳定性（例如，嗜热栖热菌蛋白酶为75℃；嗜热菌蛋白酶为65℃；胰蛋白酶为65℃；α-胰凝乳蛋白酶为45℃）。观察到孵育温度与副酪蛋白沉淀密度之间呈正相关，提高了直径测量的准确性。使用这种改良方法，可以检测到远低于其他方法检测限水平的热稳定蛋白酶（例如，在65℃下可检测到40 pg嗜热菌蛋白酶和300 pg胰蛋白酶，与传统平板测定法相比，胰蛋白酶的灵敏度提高了16倍（福苏姆，K.（1970年）《病理学与微生物学杂志》B辑78，350 - 361））。通过在凝胶中加入一些去污剂和离液剂，平板测定法的灵敏度可以进一步提高。
