Soluble Collagen Assay Kit (Fluorometric)

AgK-442

Soluble Collagen Assay Kit (Fluorometric)

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).

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.

Estimation of newly synthesized collagen in various biological samples and cultured cells

Store at -20°C.

100 assays

• Collagen Assay Buffer
• Collagenase Enzyme Mix
• Detection Reaction Buffer
• Peptide Labelling Reagent
• Developer Solution
• Collagen Standard (2 mg/ml)

Multiwell microplate spectrofluorometer
White 96-well plates with flat bottom
0.5 M acetic acid and 0.5 M sodium hydroxide (NaOH)

Detection method- Fluorescence (Ex/Em= 376/468 nm)

Mammalian soft tissues (lung, liver, etc.) homogenized in dilute acetic acid
Cultured cells (extracellular matrix of adherent cell lines) and cell culture medium (secreted extracellular collagen)