Estimation of secreted soluble collagen using K532
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April 20, 2018 -
PRLog -- Collagen is the most abundant protein in mammals, accounting for nearly 30% of body protein content. Collagen is an extracellular scaffolding protein found in skin, tendons, bone, cartilage, ligaments and muscles, amongst other tissues. There are more than twenty different characterized types of collagen, but the fibrilous types I, II and III comprise more than 90% of the total collagen in mammals. Collagen has a triple helical structure, consisting of three 'preprocollagen' strands with a repeating glycine- and proline-rich tripeptide sequence (Gly-Pro-X) and small end-cap domains called registration peptides. Nascent preprocollagen strands undergo enzymatic post-translational hydroxylation at proline and lysine residues prior to secretion to the extracellular milieu via the golgi apparatus. Once outside the cell, the dangling registration peptides are cleaved off, forming tropocollagen, the monomeric precursor to mature collagen fibers. While mature collagen exists as an insoluble web of extensively crosslinked fibrils, tropocollagen is soluble in dilute acidic solutions. Many diseases are believed to affect collagen synthesis, fibrilization and turnover, including tumor invasion and metastasis, rheumatoid arthritis, cardiopulmonary fibrosis and muscular dystrophy. BioVision's Soluble Collagen Assay Kit allows for quantification of newly synthesized (acid-soluble)
collagen levels in tissues and cultured cell medium. The assay involves extraction of soluble collagen in acetic acid, followed by enzymatic degradation of collagen into glycine-rich oligopeptides, which are quantified using a fluorogenic reagent and developer solution that selectively react with the N-terminal glycine fragments to form a stable fluorescent complex (Ex/Em = 376/468 nm). The assay is more sensitive and selective than dye-binding (Picrosirius Red) soluble collagen assays, is simple to perform and has a linear range from 0.05 – 2 μg collagen per well (2.5 μg/ml to 100 μg/ml in a 20 μl sample volume).
Figure: Estimation of secreted soluble collagen in cultured cell growth medium (DMEM/F12 medium) using BioVision's K532. 3T3-L1 fibroblasts were grown to ~80% confluence in T-75 flasks and then treated with either vehicle or TGF-β (Cat. # 6479; 100 ng/ml), a cytokine known to stimulate synthesis and secretion of tropocollagen fibrils. After 48 hours of treatment, the culture medium was removed, centrifuged to remove debris and was assayed directly (each 10 μl clarified medium, undiluted). TGF-β treatment resulted in a roughly 1.5-fold increase in collagen secretion.
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