A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Metalloproteases (MMPs) are secreted by many cells, including malignant melanoma. MMP-mediated cleavage of extracellular matrix components leads to the increased invasive potential of these cells. Gelatin zymography, presented here, is a quantifying method for studying gelatinase activity manifested as a digested gelatin area on a polyacrylamide gel.
Melanoma cells, having highly invasive properties, exhibit the formation of invadopodia—structures formed by tumor cells and responsible for the digestion of the surrounding extracellular matrix (ECM). Several metalloproteases (MMPs) are secreted by cells to hydrolyze ECM proteins. They are mainly secreted through structures known as invadopodia. ECM degradation is crucial for tumor cells while forming metastases as the cells heading towards blood vessels must loosen dense tissue.
One group of metalloproteases secreted by melanoma cells comprises the gelatinases, i.e., metalloproteases 2 and 9. Gelatinases cleave gelatin (denatured collagen), a few types of collagen (including type IV), and fibronectin, all structural components of ECM. This paper describes a gelatin zymography assay to analyze the gelatinase activity of melanoma cells. This approach is based on analyzing the extent of digestion of a substrate (gelatin) added to a polyacrylamide gel. Several advantages, such as simplicity, sensitivity, low cost, and semiquantitative analysis by densitometry, as well as the detection of both active and inactive forms of MMPs, make this assay valuable and widely used.
This protocol describes how to concentrate medium devoid of intact floating cells, cell debris, and apoptotic bodies. Next, it focuses on preparing polyacrylamide gel with gelatin addition, performing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), removing SDS, and staining of the gel to detect gelatin-free bands corresponding to the activity of gelatinases secreted by melanoma cells. Finally, the paper describes how to quantitatively analyze data from this assay. This method is a good alternative for estimating the gelatinase activity of melanoma cells to a fluorescent gelatin degradation assay, western blot, or enzyme-linked immunosorbent assays (ELISAs).
Matrix metalloproteinases (MMPs) are a family of Zn2+-containing endopeptidases that cleave various ECM proteins and non-ECM proteins, such as growth factors, cell receptors, proteinases, and their inhibitors1,2,3,4. ECM-substrate specificity of MMPs is dependent on the peptide domains and motifs, as well as similarities in their sequences, thus defining their subgroups. There are, for example, collagenases digesting various types of collagens, as well as gelatin and aggrecan; gelatinases cleaving gelatins and collagens; and matrilysins or MT-MMPs that digest various ECM proteins5.
This paper focuses on two gelatinases: MMP-2 and MMP-9, as they can digest denatured collagen (gelatin) proteolytically, allowing the detection of their activity using a gelatin zymography assay3,6. Although MMP-2 and MMP-9 bear a strong structural resemblance, they do not have identical substrate specificity7. A C-terminal hemopexin-like domain of MMPs is responsible for recognizing substrate sequence8. Slight differences in their catalytic domains are responsible for the differences in the substrate selectivity of MMP-2 and MMP-9, e.g., MMP-2, unlike MMP-9, can cleave native type I collagen9. Nevertheless, their proteolytic activities can be unquestionably determined with gelatin zymography as they both can cleave gelatin3,6.
MMPs are already known to be involved in both physiological and pathological conditions. They were found to impact cell migration, invasion, spreading, and adhesion, thus impairing angiogenesis, inflammation, tumor progression, and metastasis10,11,12,13. As they take part in various important processes, they are extensively studied due to their high therapeutic or diagnostic potential14,15,16. MMP-2 (gelatinase A) occurs as a 72 kDa proenzyme whose prodomain binds Zn2+ in the catalytic site, leading to the inhibition of enzymatic activity9. MMP-2 can be activated through the cleavage of its zymogen's prodomain by MMP-14 (MT1-MMP), thrombin, and activated protein C17,18,19,20. Therefore, the mass of active MMP-2 is lower (~64 kDa). In contrast, MMP-9 (gelatinase B) is expressed as a ~92 kDa proenzyme and is activated by the cleavage of the N-terminal domain to obtain the 83 kDa protein. MMP-9 maturation results from a prodomain cleavage by serine proteases, other MMPs, and as a response to the oxidative stress21.
The progression and malignancy of melanoma are highly dependent on the ability of the tumor cells to digest ECM, as it is "a barrier" limiting the cells from progression and metastasis formation22. Cells need to first penetrate the basal membrane (BM) to enter the dermis, migrate toward blood vessels, adhere to vascular endothelium, and reach the blood. It has been shown that gelatinase expression was increased in different cancers and was correlated with increased invasion and migration and a worse prognosis for patients23,24. MMP-2 was highly expressed in melanoma cells, with its activation state correlated with progression25,26. MMP-9 was also found to accumulate in human skin tumors and melanoma cell lines27,28.
Due to the high correlation between the properties of MMPs with the invasiveness of melanoma cells, the availability of a simple, sensitive, low-cost, functional assay to determine their presence and activity is crucial for better understanding the biology of melanoma and designing new diagnostic techniques for their detection. This paper describes the gelatin zymography technique in detail, as it can be considered the best candidate for this purpose. This approach is based on SDS-electrophoresis under denaturing but nonreducing conditions, using polyacrylamide gels prepared with the addition of gelatin29,30. Although proteins, including MMP-2 and MMP-9, are denatured in the presence of SDS during electrophoresis, washing in buffer containing Triton X-100 causes their renaturation as the result of an SDS:Triton X-100 exchange31.
These renatured MMPs digest gelatin during gel incubation in an incubation buffer, which can be finally observed as clear zymolytic bands in the Coomassie Blue-stained gel5. The amount and the area of digested gelatin presented as transparent bands corresponding to the gelatinolytic activity of MMPs can be determined using both commonly used and open-source applications—ImageLab and ImageJ29,32. Although this method has many advantages, it also possesses some limitations mentioned in the discussion. This "step by step" protocol with notes and comments performed on the different melanoma cell lines should be sufficient for reproducibility and optimization to obtain representative results. Figure 1 presents the steps of the described procedure.
1. Cell culture medium collection and concentration
2. Preparation of SDS-PAGE separating and stacking gels with gelatin (1 mg/mL) for gelatin zymography
3. Preparation of SDS-PAGE separating and stacking gels for total protein content determination
NOTE: The steps in section 3 are similar to the steps in section 2 describing gel preparation for gelatin zymography. Note that the composition of the gels is different. A gel for total protein content determination must not contain gelatin, as it will also be stained by Coomassie Brilliant Blue solution.
4. Sample loading and electrophoresis running
5. Activation, staining, and destaining of the polyacrylamide gel
6. Visualization of proteins in polyacrylamide gels
NOTE: See the Table of Materials for details on the imaging system and software used to visualize proteins in polyacrylamide gels (Figure 2A-C). Other readily available gel visualization systems (such as iBright Imaging Systems, UVP PhotoDoc-It Imaging System, E-Gel Imager, and Azure Imagers) can also be used for this purpose.
7. Data analysis
In this protocol, we describe the procedure for gelatin zymography (Figure 1) using media obtained from five melanoma cell lines (A375, SK-MEL-28, Hs-294T, WM9, WM1341D) as samples. The zymography approach shown here includes two separate SDS-PAGE electrophoreses. One SDS-PAGE electrophoresis results in the Coomassie Blue-stained gel, representing the total protein content loaded in each line (Figure 2A). It is used for the normalization of gelatinase activity d...
Despite the "step by step" protocol elaborated here, gelatin zymography requires optimization depending on the samples/cell lines being analyzed. Different cell types and cell lines (melanoma cell lines shown here) may secrete both forms (pro- and active) of MMP-2 and MMP-9 but with different gelatinase activity. The optimization of the procedure includes mainly the duration of cell starvation, the thickness of the polyacrylamide gel, the amount of loaded protein, and the duration of gel incubation in the incubat...
The authors declare no competing financial interests.
This work was supported by the National Center for Science, Poland (project #2016/22/E/NZ3/00654, granted to AJM).
Name | Company | Catalog Number | Comments |
22 µm syringe filters | Nest | 331011 | |
Acetic acid, 80% solution | Chempur | 115687330 | |
Acrylamide/bis-acrylamide, 30% Solution, 37.5:1 | Bioshop | ACR010.500 | |
Amicon Ultra-15 Centrifugal Filter Units | Millipore | UFC901008 | ultracentrifugal filter units with 10 kDa cutoff |
Ammonium Persulfate (APS) | Sigma-Aldrich | A-3678 | |
Antibiotic-Antimycotic | Gibco | 15240062 | |
Bradford reagent | Sigma | B6916 | |
Bromophenol blue | Polskie Odczynniki Chemiczne | 184070219 | |
Calcium chloride dihydrate - CaCl2 · 2H2O | Sigma-Aldrich | C-5080 | |
ChemiDoc System | Bio-rad | imaging system | |
Coomasie Brilliant Blue R-250 | Merck | 1.12553.0025 | |
Ethanol | Chempur | 1139641800 | |
FastGene Q-Stain | NIPPON Genetics EUROPE | FG-QS1 | |
Fetal Bovine Serum - FBS | Gibco | 10270-106 | |
Gelatin from porcine skin | Sigma-Aldrich | G-8150 | |
Glycerol | Sigma-Aldrich | L-4909 | |
glycine | BioShop | CAS #56-40-6 | |
high glucose Dulbecco’s modified Eagle’s medium with reduced concentration (1.5 g/l) of NaHCO3 | Pracownia Chemii Ogólnej IITD PAN | 11-500 | |
ImageJ software (Fiji) | https://imagej.nih.gov/ij/ | version 1.52p | |
ImageLab software | Bio-rad | ||
L-Glutamine | Gibco | 25030-024 | |
Mini-PROTEAN Tetra Cell | Bio-Rad | #1658001EDU | |
N,N,N′,N′-Tetramethylethylenediamine (TEMED) | Sigma-Aldrich | T9281 | |
PageRuler Prestained Protein Ladder | Thermo Fisher Scientific | 26616 | |
Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
PowerPac Basic Power Supply | Bio-rad | 1645050EDU | |
Sodium chloride - NaCl | Chempur | 7647-14-5 | |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L4509 | |
Tissue-culture 75 cm2 flask | VWR | 10062-872 | |
Trisma base | Sigma-Aldrich | T1503 | |
Triton X-100 | Sigma-Aldrich | X100 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved