一种用于检测细胞培养基中存在的蛋白水解酶的放射性标记释放微孔测定法。

A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media.

作者信息

Rucklidge G J, Milne G

机构信息

Biochemistry Division, Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom.

出版信息

Anal Biochem. 1990 Mar;185(2):265-9. doi: 10.1016/0003-2697(90)90290-p.

DOI:10.1016/0003-2697(90)90290-p

PMID:2111103

Abstract

A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.

摘要

已开发出一种改良方法，用于测量细胞培养条件培养基中的蛋白水解酶活性。该方法利用从³H标记的明胶中释放³H标记的肽，在微孔板中进行。通过戊二醛处理使底物不溶并附着于孔上，从而无需在测定结束时进行沉淀步骤。该测定方法灵敏、可重复，且适用于少量样品。可以快速评估不同蛋白酶抑制剂对活性的影响，从而对酶进行早期表征。它还可以通过用考马斯亮蓝对残留底物进行染色，适用于微孔板分光光度分析。