Home › Biochemistry › Detection of Functional Matrix Metalloproteinases by Zymography
Biochemistry JoVE (Open Access) Citable · DOI

Detection of Functional Matrix Metalloproteinases by Zymography

DOI: 10.3791/2445-v
What you'll learn
  • ✓ Prepare and run zymography gels to separate matrix metalloproteinases by activity
  • ✓ Renature and develop gels to visualize functional MMP bands
  • ✓ Analyze and interpret zymography data from biological samples
Protocol

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Difficulty
intermediate
Total time
~4–6 hours per sample batch (gel running, renaturation, and development)
Biosafety
BSL-1

Steps

1
Understand zymography principles and MMP detection

Learn the fundamentals of activity-based assays for detecting matrix metalloproteinases in culture supernatants and body fluids using zymography methodology.

▶ 00:54
2
Prepare and run zymography gel electrophoresis

Cast and load zymography gels with samples, then perform electrophoresis to separate MMPs by molecular weight under denaturing conditions.

▶ 01:23
3
Renature gels and develop enzyme activity bands

Incubate gels in renaturing buffer to restore MMP activity, then develop with substrate solution to visualize gelatinase activity as clear bands on dark background.

▶ 03:43
4
Analyze zymography results and quantify MMP activity

Document gel images and perform densitometric analysis to quantify band intensity and determine relative MMP-2 and MMP-9 activity levels.

▶ 06:07
5
Interpret representative MMP-2 detection results

Review representative zymography patterns for MMP-2, understand expected band positions, and learn to distinguish active from pro-enzyme forms.

▶ 08:03
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