Quantitative Gel Zymography Reappraised for Matrix Metalloproteinases
By LabMedica International staff writers Posted on 29 Oct 2014 |

Image: The gel zymography separation of matrix metalloproteinases (MMPs) on a 7.5% polyacrylamide gel (Photo courtesy of the Italian National Research Council).
A reappraisal of quantitative gel zymography technique for matrix metalloproteinases (MMPs) in human plasma has been used for comparison with commercially available enzyme-linked immunosorbent assay (ELISA).
The determination of MMPs is relevant in numerous pathophysiological conditions, as imbalanced MMP activity is associated with many clinical conditions, including cardiovascular diseases, especially if associated with extracellular matrix remodeling, but the results obtained are closely linked to the method used and are not directly comparable.
Scientists at the Italian National Research Council (CNR; Pisa, Italy) obtained heparinized blood samples from 25 volunteers. The samples were centrifuged and the serum stored in aliquots at −80 °C. Gel zymography was performed based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on the use of gelatin 0.5% as substrate for detection of MMP enzymatic activity. Gelatinolytic activities were detected as unstained bands against the background of Coomassie Blue stained gelatin. In order to quantify the concentrations of the analyzed protein in the unknown samples, a dose-response curve was built in each run.
Total MMP-9 was measured in diluted plasma by specific immunometric assay (R&D Systems; Minneapolis, MN, USA), previously standardized. The working range was 0.19 ng/mL to 16 ng/mL, the sensitivity was 0.05 ± 0.01 ng/mL, and the within-assay variability was 414.3 ± 23.5 ng/mL (5.7%), while the between-assay variability was less than 15%. The concentration of total MMP-9 was measured in parallel using the zymography and ELISA for comparison purposes.
The results obtained by gel zymography analysis of human blood samples using different acrylamide/bis acrylamide ratios indicated that 7.5% gels are the more suitable for visualizing MMP-9 forms. In all conditions, four bands with gelatinolytic activity are found: MMP-9 dimers, MMP-9– tissue inhibitor of metalloproteinase1 (TIMP-1) complex, MMP-9, and MMP-2. The time course of the proteolytic activity of MMPs indicated 16 to 24 hours as optimal incubation time and these optimal parameters were used for routine assay. A linear positive correlation was found between the MMP-9 values of zymography and ELISA with the ELISA values significantly lower than those obtained with zymography.
The authors concluded that the main advantage of gel zymography is that it allows visualization of both the latent and active forms of gelatinases. The presence of MMP dimers and MMP/TIMP complexes in the plasma samples, revealed by gel zymography, could thoroughly characterize the role of MMP in associated diseases. Gel zymography allows separation and visualization of MMP-2 bands, so both MMP-2 and MMP-9 can be determined in the same run from a single biological sample. The study was published in the September 2014 issue of the Journal of Clinical Laboratory Analysis.
Related Links:
Italian National Research Council
R&D Systems
The determination of MMPs is relevant in numerous pathophysiological conditions, as imbalanced MMP activity is associated with many clinical conditions, including cardiovascular diseases, especially if associated with extracellular matrix remodeling, but the results obtained are closely linked to the method used and are not directly comparable.
Scientists at the Italian National Research Council (CNR; Pisa, Italy) obtained heparinized blood samples from 25 volunteers. The samples were centrifuged and the serum stored in aliquots at −80 °C. Gel zymography was performed based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on the use of gelatin 0.5% as substrate for detection of MMP enzymatic activity. Gelatinolytic activities were detected as unstained bands against the background of Coomassie Blue stained gelatin. In order to quantify the concentrations of the analyzed protein in the unknown samples, a dose-response curve was built in each run.
Total MMP-9 was measured in diluted plasma by specific immunometric assay (R&D Systems; Minneapolis, MN, USA), previously standardized. The working range was 0.19 ng/mL to 16 ng/mL, the sensitivity was 0.05 ± 0.01 ng/mL, and the within-assay variability was 414.3 ± 23.5 ng/mL (5.7%), while the between-assay variability was less than 15%. The concentration of total MMP-9 was measured in parallel using the zymography and ELISA for comparison purposes.
The results obtained by gel zymography analysis of human blood samples using different acrylamide/bis acrylamide ratios indicated that 7.5% gels are the more suitable for visualizing MMP-9 forms. In all conditions, four bands with gelatinolytic activity are found: MMP-9 dimers, MMP-9– tissue inhibitor of metalloproteinase1 (TIMP-1) complex, MMP-9, and MMP-2. The time course of the proteolytic activity of MMPs indicated 16 to 24 hours as optimal incubation time and these optimal parameters were used for routine assay. A linear positive correlation was found between the MMP-9 values of zymography and ELISA with the ELISA values significantly lower than those obtained with zymography.
The authors concluded that the main advantage of gel zymography is that it allows visualization of both the latent and active forms of gelatinases. The presence of MMP dimers and MMP/TIMP complexes in the plasma samples, revealed by gel zymography, could thoroughly characterize the role of MMP in associated diseases. Gel zymography allows separation and visualization of MMP-2 bands, so both MMP-2 and MMP-9 can be determined in the same run from a single biological sample. The study was published in the September 2014 issue of the Journal of Clinical Laboratory Analysis.
Related Links:
Italian National Research Council
R&D Systems
Latest Clinical Chem. News
- New Clinical Chemistry Analyzer Designed to Meet Growing Demands of Modern Labs
- New Reference Measurement Procedure Standardizes Nucleic Acid Amplification Test Results
- Pen-Like Tool Quickly and Non-Invasively Detects Opioids from Skin
- Simple Urine Test Could Detect Multiple Cancers at Early Stage
- Earwax Test Accurately Detects Parkinson’s by Identifying Odor Molecules
- First-Of-Its-Kind Quantitative Method Assesses Opioid Exposure in Newborns
- Paper-Based Devices Outperform Existing Methods in Diagnosing Asymptomatic Malaria
- Simple Skin Test Could Revolutionize Diagnosis of Pediatric Eosinophilic Esophagitis
- Portable Diagnostic Tool Uses Bioluminescence to Detect Viruses at POC
- AI-Powered Lung Maturity Test Identifies Newborns at Higher Risk of Respiratory Distress
- AI-Powered Blood Test Accurately Detects Ovarian Cancer
- Automated Decentralized cfDNA NGS Assay Identifies Alterations in Advanced Solid Tumors
- Mass Spectrometry Detects Bacteria Without Time-Consuming Isolation and Multiplication
- First Comprehensive Syphilis Test to Definitively Diagnose Active Infection In 10 Minutes
- Mass Spectrometry-Based Monitoring Technique to Predict and Identify Early Myeloma Relapse
- ‘Brilliantly Luminous’ Nanoscale Chemical Tool to Improve Disease Detection
Channels
Molecular Diagnostics
view channel
Blood-Based Biomarkers Could Detect Alzheimer's as Early as Middle Age
As the global population ages, Alzheimer's disease and other dementing diseases are becoming more prevalent. The disease processes leading to Alzheimer's symptoms can begin years or even decades before... Read more
RNA Screening Test Could Detect Colon Polyps Before They Become Cancerous
Colorectal cancer has become a growing health crisis, especially as it increasingly affects younger adults in their 20s, 30s, and 40s, while screening rates remain low. Colorectal cancer is now the leading... Read more
New RT-LAMP Assay Offers Affordable and Reliable Pathogen Detection for Resource-Limited Settings
The high cost and logistical complexities associated with rapid, point-of-care tests have long hampered widespread access to molecular diagnostics, especially in low- and middle-income countries.... Read moreHematology
view channel
Disposable Cartridge-Based Test Delivers Rapid and Accurate CBC Results
Complete Blood Count (CBC) is one of the most commonly ordered lab tests, crucial for diagnosing diseases, monitoring therapies, and conducting routine health screenings. However, more than 90% of physician... Read more
First Point-of-Care Heparin Monitoring Test Provides Results in Under 15 Minutes
Heparin dosing requires careful management to avoid both bleeding and clotting complications. In high-risk situations like extracorporeal membrane oxygenation (ECMO), mortality rates can reach about 50%,... Read moreImmunology
view channel
Evolutionary Clinical Trial to Identify Novel Biomarker-Driven Therapies for Metastatic Breast Cancer
Metastatic breast cancer, which occurs when cancer spreads from the breast to other parts of the body, is one of the most difficult cancers to treat. Nearly 90% of patients with metastatic cancer will... Read more
Groundbreaking Lateral Flow Test Quantifies Nucleosomes in Whole Venous Blood in Minutes
Diagnosing immune disruptions quickly and accurately is crucial in conditions such as sepsis, where timely intervention is critical for patient survival. Traditional testing methods can be slow, expensive,... Read moreMicrobiology
view channel
Viral Load Tests Can Help Predict Mpox Severity
Mpox is a viral infection that causes flu-like symptoms and a characteristic rash, which evolves significantly over time and varies between patients. The disease spreads mainly through direct contact with... Read more
Gut Microbiota Analysis Enables Early and Non-Invasive Detection of Gestational Diabetes
Gestational diabetes mellitus is a common metabolic disorder marked by abnormal glucose metabolism during pregnancy, typically emerging in the mid to late stages. It significantly heightens the risk of... Read morePathology
view channel
Novel Method Tracks Cancer Treatment in Cells Without Dyes or Labels
Multiple myeloma is a blood cancer that affects plasma cells in the bone marrow, leading to abnormal protein production, weakened immunity, and organ damage. Traditional methods for evaluating myeloma... Read more
New AI-Based Method Effectively Identifies Disease Phenotypes Using Light-Based Imaging
Precision medicine, where treatment strategies are tailored to a patient's unique disease characteristics, holds great promise for cancer therapy. However, identifying disease phenotypes, which are critical... Read more
AI Accurately Predicts Genetic Mutations from Routine Pathology Slides for Faster Cancer Care
Current cancer treatment decisions are often guided by genetic testing, which can be expensive, time-consuming, and not always available at leading hospitals. For patients with lung adenocarcinoma, a critical... Read moreTechnology
view channel
Multifunctional Nanomaterial Simultaneously Performs Cancer Diagnosis, Treatment, and Immune Activation
Cancer treatments, including surgery, radiation therapy, and chemotherapy, have significant limitations. These treatments not only target cancerous areas but also damage healthy tissues, causing side effects... Read more
Ultra-Sensitive Biosensor Based on Light and AI Enables Early Cancer Diagnosis
Cancer diagnosis is often delayed due to the difficulty in detecting early-stage cancer markers. In particular, the concentration of methylated DNA in the bloodstream during the early stages of cancer... Read moreIndustry
view channel
BD Biosciences & Diagnostic Solutions to Merge with Waters
BD (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and Waters Corporation (Milford, MA, USA) have entered into a definitive agreement to combine BD's Biosciences & Diagnostic Solutions... Read more