Gelatin zymography protocol

Last edited Thu 24 June 2025

In this procedure, active gelatinases digest gelatin embedded in a polyacrylamide gel; other substrates such as casein or laminin can also be used depending on the target protease. After Coomassie staining, areas of degradation are visible as clear bands against a darkly stained background.

Gelatin zymography is a sensitive technique capable of detecting low levels of MMP activity.

This protocol is optimized for detecting secreted MMP-9 and MMP-2 activity in conditioned media. The technique allows for the detection and distinction of both pro- and active forms of MMPs, enabling assessment of active enzyme levels.

Introduction to Gelatin Zymography

Gelatin zymography is a highly sensitive enzyme assay technique designed to detect and characterize proteolytic enzymes, especially matrix metalloproteinases (MMPs). This method utilizes a polyacrylamide gel embedded with a suitable substrate, such as gelatin, to specifically monitor protease activity. As MMPs play a crucial role in the degradation of extracellular matrix components, gelatin zymography has become an essential tool for studying MMPs in various biological and pathological contexts. The technique allows researchers to visualize the activity of these proteolytic enzymes directly within the gel, making it possible to analyze even low levels of enzyme activity. Because of its sensitivity and specificity, gelatin zymography is widely used in research areas such as cancer, arthritis, and inflammation, where understanding the dynamics of MMPs and their impact on tissue remodeling is vital.

Stage 1 - Preparation of conditioned media

Materials required

• Your cells
• Six-well plates or flasks
• FBS-free media
• Centrifuge

Steps

Plate cells cultured in fetal bovine serum (FBS).

• We can plate cells in a six-well plate (2 mL/well) or in a 75 cm² flask (10 mL).

At 70–80% confluency, remove FBS media.

• Wash cells twice with FBS-free media and continue to grow cells in FBS-free media.

Collect conditioned media and centrifuge or filter to eliminate dead cells.

Concentrate conditioned media 10X.

Gel Preparation

Proper gel preparation is a critical step in the success of gelatin zymography. The process involves creating both a separating gel and a stacking gel. The separating gel, which contains the gelatin solution as a substrate, is responsible for resolving proteins based on their molecular weight, allowing for the clear identification of proteolytic activity. The stacking gel, meanwhile, serves to concentrate the proteins into a tight band at the top of the gel, ensuring sharp separation during electrophoresis. To prepare the gel, the gelatin solution is thoroughly mixed with acrylamide/bis-acrylamide, followed by the addition of ammonium persulfate and TEMED to initiate polymerization. The resulting mixture is poured into a gel cassette and allowed to set, forming a stable matrix for the subsequent enzyme assay. Careful attention to this step ensures that the substrate is evenly distributed and that the gel is properly polymerized, which is essential for reliable and reproducible results in gelatin zymography.

Stage 2 - Gelatin zymography: running the gel for MMPs

Materials required

• Separating gel (7.5% acrylamide): 2 mL of 1.5 M Tris pH 8.8, 2 mL of 30% acrylamide, 2 mL of H₂O, 2 mL of 4 mg/mL gelatin, 80 μL of 10% SDS, 80 μL of 10% APS, 10 μL of TEMED.
• Stacking gel: 1.25 mL of 0.5 M Tris pH 6.8, 0.67 mL of 30% acrylamide, 3.08 mL of H₂O, 50 μL of 10%SDS, 50 μL of 10% APS, 10 μL of TEMED.
• 5X non-reducing sample buffer (pH 6.8): 4% SDS, 20% glycerol, 0.01% bromophenol blue, 125 mM Tris-HCl, H₂O.

Recipe to prepare 250 mL of 5X non-reducing sample buffer:

Steps

Dilute conditioned media so that all samples have the same protein concentration.

• For each sample, test one aliquot at a low protein concentration (5 µg/mL) and one at a high protein concentration (15 µg/mL).

Add 5X non-reducing sample buffer to your samples.

Prepare a 7.5% acrylamide gel containing gelatin.

• Use a 1 mm thickness plate.

Load sample to each well.

• Typically, 10 μL protein per well is suitable.

Run the gel at 150 V until good band separation is achieved.

Stage 3 - Gel washing and staining

Materials required

• Washing buffer (pH 7.5)

• Incubation buffer (pH 7.5)

• Staining solution, 100 mL: 40 mL of methanol, 10 mL of acetic acid, 50 mL of H₂O, and 0.5 g of Coomassie Blue)
• Destaining solution, 1 L: 400 mL of methanol, 100 mL of acetic acid, 500 mL of H₂O.

Steps

Wash the gel 2 x 30 min with the washing buffer at room temperature with agitation.

Rinse for 5–10 min in incubation buffer at 37°C with agitation.

Replace with fresh incubation buffer and incubate for 24 h at 37°C.

Stain the gel with staining solution for 30 min to 1 h at room temperature with agitation.

Rinse with water 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.

Analyzing the zymogram

After incubation and staining, areas where protease activity has occurred will appear as clear bands, as the digested gelatin no longer retains the dye. By comparing these clear bands to molecular weight standards run on the same gel, you can determine the molecular weight of the active proteases present in the sample. The intensity and sharpness of the clear bands provide a direct measure of protease activity, enabling quantitative and qualitative analysis of enzyme function. This visual representation makes it easy to assess differences in protease activity across samples, cell types, or experimental conditions.

Troubleshooting common issues

Despite its robustness, gelatin zymography can present several challenges, such as poor gel resolution, unexpectedly low protease activity, or interference from bacterial proteases. To address these issues, it is important to optimize gel preparation and running conditions, ensuring that the gel is properly polymerized and that sample preparation is consistent.

Including positive controls in each run can help verify that the assay is functioning correctly. The use of selective inhibitors can aid in identifying specific proteases and distinguishing them from background activity.

If bacterial proteases are suspected, steps should be taken to minimize bacterial contamination during sample collection and handling.

Additionally, reverse zymography can be employed to detect tissue inhibitors of metalloproteinases (TIMPs), providing insight into the balance between protease activity and inhibition.

Careful attention to these troubleshooting strategies will help ensure reliable and interpretable results from your gelatin zymography experiments.

Applications of gelatin zymography

Gelatin zymography is a versatile technique with broad applications. It is widely used to investigate protease activity in physiological and pathological conditions, including tumor progression, inflammation, and tissue remodeling. Researchers can apply this method to analyze proteolytic enzymes in a variety of biological samples, such as tissue extracts, cell lines, and culture media. The technique is also valuable for evaluating the effectiveness of protease inhibitors and for identifying potential therapeutic targets in diseases characterized by abnormal protease activity. By providing detailed information about the presence and activity of specific enzymes, gelatin zymography supports studies on the regulation of extracellular matrix components and the role of proteases in health and disease. Its ability to deliver sensitive, quantitative, and qualitative data makes it an indispensable tool for exploring the complex dynamics of proteolytic enzymes in diverse biological systems.

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