Reverse zymography (substrate gel electrophoresis) kit for detection of TIMP activities.
The kit provides reagents for detection of TIMPs using a gel activity assay system. It can detect TIMP proteins from a wide range of species including human, mouse, rat, horse, dog and chick.
A research team at the University of East Anglia has generated a versatile and easy to use kit that allows the detection of TIMP (tissue inhibitor of metalloproteinase) proteins using an SDS-polyacrylamide gel electrophoresis (SDS-PAGE)-based activity assay. It includes a source of matrix metalloproteinase MMP gelatinase activity and TIMP standards. The method works optimally with samples from cultured cells, but it can also be used for analysis of other biological samples.
The kit contains a calibrated solution of gelatinase-A (MMP-2) that is added to the SDS-PAGE gel mixture before casting the gel. A set amount of gelatin is also added. The samples are run on the gel in a standard fashion but without heat treatment or reduction prior to loading. After electrophoresis, the gel is washed to remove SDS and then is incubated in buffer that allows the gelatinase to digest the gelatin, except where it is inhibited by TIMP proteins. After staining with Coomassie blue, the result is a gel with a pale blue background (where gelatin was degraded by the gelatinase) with blue bands showing the positions and relative amounts of TIMPs. TIMP-1, -2, and -3 can be distinguished by virtue of their differential mobility on SDS-PAGE.
The MMP solution is provided in ten 1ml aliquots, sufficient for carrying out ten standard 18×12 cm reverse zymograms. In addition, the kit contains samples of TIMP-1, -2, and -3 standards to allow verification of the procedure.
Free TIMP-1, -2 and -3 can be readily identified in a sample without the use of antibodies. The same reagents can detect TIMPs from various species without modification of the procedure. The method is sensitive, and with calibration can be made quantitative.