Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

Abstract

Substantive scientific and intellectual contributions to the study, responsible for conception and design, interpretation of data, manuscript writing, critical revision, final approval of the version to be published. ABSTRACT PURPOSE:To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam 3 Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS:To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography.RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION:Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.Key words: Zymography. Matrix Metalloproteinase 2. Fibroblasts. Periodontal Ligament. Toll-Like Receptors. Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

Early Regenerative Modifications of Human Postmenopausal Atrophic Vaginal Mucosa Following Fractional CO2 Laser Treatment

“…In this paper, early structural modifications of atrophic vaginal mucosa in postmenopausal women after a very short time (1 hour after one fractional CO 2 application) have been studied, to identify the structures as the first involved in the starting of regenerative mechanisms. Also, we have also considered the role of metalloproteinase MMP - 2, well known as an interstitial collagenase [ 16 ], by zymography technique, an efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate [ 17 ]. The degree of digestion of the substrate into the zymograms by purified gelatinase - A (MMP - 2, the matrix metalloprotease of interest extracted from bioptic samples) is proportional to the enzyme loading, and varying the incubation time results in a shift in the linear range of the assay [ 18 ].…”

A Bifunctional Molecule with Lectin and Protease Inhibitor Activities Isolated from Crataeva tapia Bark Significantly Affects Cocultures of Mesenchymal Stem Cells and Glioblastoma Cells

“…Gelatinolytic activity of metalloproteases appears as a translucent band with a blue background. In the images, the color scale was inverted to white/black and the band intensity was analyzed by densitometry method with ImageJ software [43].…”

Zymography-types, advantages, uses and troubleshooting - A detailed review

“…Several methods for studying the expressions and behaviours of MMPs have been published in the literature like IHC, ELISA etc. Zymography is an important approach that uses SDS-PAGE gels copolymerized with a protein substrate to electrophorese (Lisboa et al, 2013).…”

“…MMP-2 and MMP-9 proteolytic activity is measured using gelatin as a protein substrate, casein for MMP-3, MMP-7 and MMP-10, and collagen for MMP-1. Zymography enables the active and latent types of MMPs to be identi ied in a composite mixture such as cultured media or iltered preparations depending on molecular weight (Lisboa et al, 2013).…”

“…When cultures were co-activated with only TLRs 2 and 4 ligands or under mechanical force treatment, MMP-2 activity increased relative to baseline levels. Hence, it is clear that zymography is a very effective process for studying MMP activities (Lisboa et al, 2013). See Figure 6.…”