Description
The Biocolor Sircol™ INSOLUBLE Collagen Assay Kit is a dye-binding assay designed for accurate quantification and measurement of insoluble or cross-linked collagen fibers. It is ideal for analyzing materials from sources such as tissues, bone, and calcified tissue.
The assay can be used to assess the rate of production of newly laid down collagen fibres during periods of rapid growth, development, tissue repair, remodeling and wound healing.
- It replaces the conventional hydroxyproline analysis. No more working with concentrated acids or alkalis!
- The Sircol Insoluble Assay uses mild acid and temperature treatment for 2 to 3 hours.
- The solubility conversion to denatured collagen and subsequent measurement can be completed within one working day.
- The ratio of soluble and insoluble collagens can be measured using the same Sircol Dye Reagent.
Principle of Method
Sircol™ Dye Reagent contains Sirius Red - a linear anionic dye with sulphonic acid side chain groups. Under assay conditions the Sircol dye binds the basic groups of soluble collagen molecules. Maximal binding occurs in collagens possessing intact triple helix organization as the highly ordered Gly-X-Yn helical structure of tropocollagen further contributes to dye binding. This results in a high degree of dye-collagen specificity. Affinity is progressively reduced during heat denaturation 4ºC due to the unwinding of the triple helix and formation of random chains.
Step 1. Samples being assayed for insoluble collagen must first undergo a 2-3 hour pre-treatment with Sircol Fragmentation reagent. This converts insoluble collagen into water-soluble gelatin can then be assayed.
Step 2. Addition of Sircol Dye Reagent to these pre-treated insoluble collagen samples results in the formation of a denatured collagen-dye complex. This complex then precipitates during the dye incubation period and is subsequently isolated by centrifugation, followed by washing to remove unbound dye. The Denatured collagen-bound dye is then eluted and measured spectrophotometrically.
Step 3. The insoluble collagen content of unknown samples is quantified by comparison against a calibration curve prepared using a the denatured collagen standard supplied with the kit.
Specifications
| Detection Method | Colorimetric Detection (556 nm) |
| Assay Range | 0 - 1000 µg/ml |
| Limit of Detection | 100 µg/ml |
| Assay Run-Time | 4 hours (including conversion of native insoluble collagen to soluble denatured collagen) |
| Type of Collagen Measured | Covalent crosslinked insoluble collagen |
| Measurements Per Kit | 110 in total (allows a maximum of 46 samples to be run in duplicate alongside a standard curve). |
Kit Contents
The standard size Sircol™ Insoluble Collagen Assay kit contains:
- Sircol Dye Reagent (1x 110 ml)
- Denatured Collagen Reference Standard (1x 5 ml, 1.0 mg/ml)
- Acid-Salt Wash Reagent (1x 20 ml)
- Fragmentation Reagent (1x 110 ml)
- Alkali Reagent (1x 110 ml)
- 2 ml screw-cap tubes for preparation of samples.
- Assay kit manual
Application Notes
Insoluble collagens must be converted into soluble form prior to assay. Instructions and reagents are provided with the kit. Depending on the sample, this will require prior salt/acid/acid-pepsin extraction.
Non-mammalian collagens may result in a reduced limit of detection. We recommend use of an assay standard matched to the non-mammalian species under assay.
Which Sircol Collagen Assay should I use for my application?
Resources
- Product Datasheet (PDS)
- Material Safety Data Sheet (MSDS)
- Product References
- Frequently Asked Questions (FAQ)
Want to learn more about collagen? We have compiled the links below that contain information which you may find interesting.
- The Collagen Family
- Collagen In Wound Healing
- Collagen Assembly at the Cell Surface: Dogmas Revisited
- Natural Biomaterials and Their Use as Bioinks for Printing Tissues
- Additive Biomanufacturing with Collagen Inks
- Collagen Mimetic Peptides
- The role of collagen in cancer: from bench to bedside
- Collagen of Extracellular Matrix from Marine Invertebrates and Its Medical Applications
Related Products
- Biocolor Sircol™ 2.0 Soluble Collagen Assay (96-well plate format)
- Biocolor Sircol™ Soluble Collagen Assay (original version)
- Collagen and Hydroxyproline Assays
- Extracellular Matrix Assays
- Extracellular Matrix Molecules
- Promed Bioscience high-purity collagen biomaterials
Ilex Life Sciences LLC is an official distributor of Biocolor products.