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JoVE Encyclopedia of Experiments
Encyclopedia of Experiments: Biological Techniques

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Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity

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Matrix metalloproteinase, or MMP, a multi-domain protease comprising propeptide and catalytic domains, is secreted as an inactive enzyme or zymogen by specific cells. The interaction between the catalytic domain zinc ion and the propeptide domain cysteine residue keeps the MMP inactive.

To detect MMPs in cell culture supernatant by zymography gel electrophoresis, fill the electrophoresis tank containing gel cassette with sodium dodecyl sulfate or SDS-containing running buffer.

Load the culture supernatant mixed with loading buffer, followed by protein standards, into wells of polyacrylamide gel, copolymerized with an MMP-specific protein substrate.

SDS, a negatively-charged detergent, under non-reducing conditions, disrupts the cysteine-zinc interactions in MMPs and denatures the proteins while imparting a negative charge.

Perform electrophoresis. The electric field causes the negatively charged proteins to migrate toward the positively charged anode, causing size-based separation of proteins.

Post electrophoresis, treat the gel with a non-ionic detergent-containing renaturing buffer. Non-ionic detergents replace SDS, renaturing proteins and partially restoring the MMP activity. MMPs further get processed to a fully active state.

Incubate in a developing buffer containing divalent metal cations essential for MMP activity. Active MMPs, along with cations, digest the MMP substrate in the gel. Wash the gel to remove the digested products. Stain the gel.

The areas of MMP activity, devoid of MMP substrate, appear as clear bands against a dark blue background of undigested substrate. Using suitable software, the gel band intensity can be correlated to MMP activity.

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