What is gelatin Zymography?
Gelatin zymography is mainly used for the detection of the gelatinases, MMP-2 and MMP-9, respectively (Figure 2). It is extremely sensitive because levels of 10 pg of MMP-2 can already be detected (43). It should be considered, however, that other MMPs, such as MMP-1, MMP-8, and MMP-13 can also lyse the substrate.
What is Zymogram analysis?
Zymography describes the experimental technique in which an enzyme activity is analyzed in situ following electrophoresis. It allows characterization of important physical properties of the enzyme within crude cell extracts or partially purified fractions.
What is in situ zymography?
In situ zymography is a unique laboratory technique that enables the localisation of matrix-degrading metalloproteinase (MMP) activity in histological sections. Frozen sections are placed on glass slides coated with fluorescently labelled matrix proteins.
How are MMPS activated?
The MMP activation by reactive oxygen is driven through preferential oxidation of the thiol–zinc interaction and autocatalytic cleavage, followed by enzyme inactivation with extended exposure by modification of amino acids critical for catalytic activity, as shown in vitro for MMP-7 [30].
What is the purpose of zymography?
Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful, but often misinterpreted, tool yielding information on potential hydrolytic activities, enzyme forms and the locations of active enzymes.
What is zymography used for and what is its principle?
Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period.
How are MMPs secreted?
MMPs are secreted as proenzymes, which are activated by proteolytic cleavage and regulated by a family of inhibitors called the tissue inhibitors of matrix metalloproteinases (TIMPs), which are constitutively produced by a variety of cells.
What do metalloproteinases do?
A member of a group of enzymes that can break down proteins, such as collagen, that are normally found in the spaces between cells in tissues (i.e., extracellular matrix proteins). Because these enzymes need zinc or calcium atoms to work properly, they are called metalloproteinases.
What are different types of in gel zymography?
2.2. Gel Zymography Reagents and Solutions
- Resolving Gel. 1.5 M Tris-Cl, pH 8.8.
- Stacking Gel. 0.5 M Tris-Cl with 0.4% SDS, pH 6.8.
- 10× Electrophoresis Running Buffer. 30.3 g Tris-base.
- 5× Sample Buffer. 0.313 M Tris–HCl, pH 6.8.
- Gel Washing Buffer.
- Gel Development Buffer.
- Gel Staining Solution.
- Gel Destaining Solution.
What is in vivo zymography?
The zebrafish in particular, with its transparent, rapidly and externally developing embryo, provides an ideal experimental model for the application of this technique (dubbed ‘in vivo zymography’ or IVZ), because it allows for the high-resolution imaging of the fluorescent degradation products of the introduced …
Where are MMPs produced?
MMPs are produced by many cell types, including lymphocytes and granulocytes, but in particular by activated macrophages (17).
What activates MMPs?
What is gelatin zymography and how is it used?
Gelatin zymography is an extremely sensitive and useful technique for measuring the relative amounts of active and inactive gelatinase (MMP-2 or MMP-9) in samples.
How do you stain gelatin for zymography?
Gelatin zymography. Stain the gel with staining solution for 30 min to 1 h at room temperature with agitation. Rinse with H 2 O until excess staining solution is removed. Incubate with destaining solution until bands can clearly be seen. Areas of enzyme activity appear as white bands against a dark blue background.
How do you prepare a zymogram for gel electrophoresis?
Mix at least 15 uL of each samples 1:1 (vol:vol) with at least 15 uL of Sample Buffer (2X) and allow to sit 5 minutes at room temperature. Remove the gelatin zymogram from its package and rinse it with distilled water Assemble the electrophoresis chamber with the zymogram according to manufacturer’s instructions.
How can I reduce gel-to-gel variations in my zymograms?
To reduce the effect of gel-to-gel variations, it’s best to normalize the MMP activity of your samples to an active MMP-2 standard (fully explained in our video: Analyzing Gelatin Zymograms Part 2). Click here to see our protocol on Preparing MMP-2 Standards.