What is casein zymography?
What is casein zymography?
Casein zymography is used to detect calpain activity in cell or tissue lysates. In this technique, lysates are loaded into a polyacrylamide gel containing casein, and the enzymes within the lysates are electrophoretically separated.
How do I find my MMP activity?
In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs …
What is MMP gene?
Matrix metalloproteinases (MMPs), also known as matrix metallopeptidases or matrixins, are metalloproteinases that are calcium-dependent zinc-containing endopeptidases; other family members are adamalysins, serralysins, and astacins. The MMPs belong to a larger family of proteases known as the metzincin superfamily.
How do you do zymography?
- Dilute conditioned media so that all samples have the same protein concentration.
- Add 5X non-reducing sample buffer to your samples.
- Prepare a 7.5% acrylamide gel containing gelatin.
- Load sample to each well; typically 10 μL protein per well is suitable.
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 .
What does MMP 9 do?
Matrix metalloproteinase (MMP)-9, one of the most widely investigated MMPs, regulates pathological remodeling processes that involve inflammation and fibrosis in cardiovascular disease. MMP-9 directly degrades extracellular matrix (ECM) proteins and activates cytokines and chemokines to regulate tissue remodeling.
Is MMP a gene?
MMPs are expressed as zinc-dependent endopeptidases and have a wide spectrum of biological substrates that are overlapping. There are 24 genes encoding MMPs in humans, including duplicated MMP-23 gene. So, there are 23 different MMPs in humans .
What is zymography used for?
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.
What do MMPs cleave?
MMPs may be described as multifunctional enzymes capable of cleaving the extracellular matrix components (collagens, laminin, fibronectin, vitronectin, aggrecan, enactin, versican, perlecan, tenascin, elastin and many others), growth factors, cytokines and cell surface-associated adhesion and signaling receptors.
How can I decrease my MMP-9?
EPA and DHA significantly decreased MMP-9 protein levels, MMP-9 activity, and significantly inhibited human T cell migration. The data suggest that omega-3 fatty acids may benefit patients with multiple sclerosis by modulating immune cell production of MMP-9.