Determination of the activity of the substring protease.

：

MMP-2MMP-9,,MMP-1、MMP-3,？！
：,。
：MMP-1（Collagenases）
MMP-2MMP-9（Gelatinases）
MMP-3（Stromelysins）
,MMP-1, MMP-2 and MMP-9,MMP-3,。 ：
MMP enzyme expression was assayed by
SDS
-PAGE zymography using either gelatin or casein as MMP substrates. For gelatin-containing zymograms, equal volumes (10 ul) of samples　normalized for protein concentration were subjected to electrophoresis, without boiling or reduction, through a 10% polyacrylamide gel co-polymerized with gelatin (0.5 mg/ml) at 4℃. For casein-containing zymograms, the samples (20 ul each) were subjected to electrophoresis through a 4–16% polyacrylamide gel containing blue-stained beta-casein (0.5 mg/ml)(Novex). After electrophoresis was complete, the gel was incubated for 1 hour at 25℃ in a 2.5% Triton X-100 solution, washed two times, 20 minutes each, with water and then incubated overnight at 37℃ in a 0.05 M Tris-HCl buffer, pH 8.0, containing 5 mM CaCl2. As a control, duplicate samples were loaded onto another gel that was then incubated in a 0.05 M Tris-HCl buffer, pH 8.0, containing 10 mM
EDTA
to inhibit MMP activity. The gels were fixed with 40% methanol and 7% acetic acid, stained with 0.25% Coomassie brilliant blue R250 and then destained with 10% methanol and 7% acetic acid. Enzyme activity attributed to MMP-1, MMP-2 and MMP-9 can be visualized in the gelatin-containing zymograms as clear bands against a blue background. Standards for the active forms of recombinant humanMMP-2 and MMP-9 were included on the gels for comparison and identification. Similarly, casein-containing zymograms were used to determine MMP-3 activity. Relative clearing of each sample was quantitated by determining the inverse optical density units using the NIH Image version 1.60 software package.
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