Gelatin Degradation Assay Kit (Alternative to Zymography) (ab234057)

Overview

  • Product name

    Gelatin Degradation Assay Kit (Alternative to Zymography)
  • Detection method

    Fluorescent
  • Sample type

    Tissue, Cell Lysate
  • Assay type

    Quantitative
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    Gelatin Degradation Assay Kit (Alternative to Zymography) (ab234057) utilizes a hybrid approach for the detection of gelatinase activity by employing a highly quenched gelatin substrate which upon cleavage by a suitable gelatinase releases a fluorophore, which can be easily quantified using a conventional microplate reader. This method is substrate-specific, simple, fast, high-throughput adaptable and amenable to the sensitive detection of gelatinase activity (as low as 0.06 mCDU for bacterial collagenase) in biological samples.

  • Notes

    Gelatinases are a type of matrix zinc-dependent metalloproteases (MMPs) that degrade gelatins and a variety of other extracellular matrix proteins. These enzymes are synthesized as latent zymogens that are activated by proteolysis and inhibited by tissue inhibitors of metalloproteases (TIMPs). Two mammalian gelatinases, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are critical for basement membrane degradation and are highly upregulated in variety of tumor cells. Gelatinase activity is usually detected by small peptide-based activity assays which may suffer from lack of substrate specificity. Other methods for gelatinase activity include gelatin Zymography where samples are electrophoresed on a gelatin-containing SDS-PAGE, and further renatured in a suitable buffer for 12-16 h. The zymogram is subsequently stained, and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme. Such methods are laborious, time-consuming and may lead to the loss of enzymatic activity as renaturation may not be completely reversible.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    Cell Lysis Buffer 1 x 25ml
    Enzyme Positive Control 1 x 10µl
    FITC Standard (5 mM) 1 x 10µl
    Gelatinase Assay Buffer 1 x 25ml
    Gelatinase Substrate (FITC) 1 vial
  • Relevance

    Gelatin is denatured collagen composed of heterogeneous mixture of water-soluble proteins of high average molecular weights. Below 35-40°C gelatin swells and absorbs 5-10 times its weight of water to form a gel. Gel strength and viscosity gradually weaken upon prolonged heating in solution above 40°C.

Images

Protocols

References

ab234057 has not yet been referenced specifically in any publications.

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