What is Collagen?

Collagen is a fundamental component of the extracellular matrix, and the predominant protein in animals, constituting around 30% of total protein mass. A glycoprotein, it is well known for its triple helical structure. This is formed from three polypeptide α-chains with Gly-X-Y repeating residues (Gly for Glycine, X for proline, and Y for hydroxyproline).

‍Types of Collagen

Over 28 types of collagens have been identified, with Type I collagen being the most abundant. It’s prevalent in ligaments, tendons, skin, and bone tissue. Its mature, insoluble form grants it remarkable strength, making it vital for the mobility of organisms. Collagen also has biochemical functions, influencing cell growth, proliferation, and differentiation.

This version of the kit is designed to detect and measure  SOLUBLE forms of collagen. Chose the Sircol Insoluble collagen kit if you need to analyse INSOLUBLE collagen.

Applications of Collagen

Collagen, with its diverse properties, finds utility in various industries. It plays a role in medicine for wound healing and has an expanding role in tissue engineering and cell culture for biomedical purposes. It’s gaining popularity in the cosmetic industry for skin rejuvenation and is used in chemical formulations and the food industry as a functional food supplement and additive.

How does Sircol 2.0 detect collagen?

The Sircol 2.0 dye reagent includes Sirius Red, a linear anionic dye with sulfonic acid side chains. This reagent is specially formulated to bind to the Gly-X-Yn helical structure of soluble collagen under assay conditions.

*The improved formulation of Sircol 2.0 dye enables a greater degree of dye-collagen specificity (compared to our previous S1000 assay kit).

‍Overview of the Sircol 2.0 assay process:

Step 1. Prepared samples are placed in the wells of the assay microplate, together with Sircol Dye Reagent. After 30 minutes mixing, any collagen-dye complexes will form as a precipitate. These are collected on the base of the microplate wells by centrifugation.

Step 2. Unbound dye is removed by gentle aspiration, followed by a rinse with Plate Wash Reagent.

Step 3. Following further centrifugation, collagen-bound dye is eluted by incubation with a Dye Release Reagent. Eluted dye is detected ‘in-situ’ by spectrophotometric analysis of the microplate at 556nm.

Step 4. The collagen content of unknown samples can be quantified by comparison against a calibration curve, prepared using the Collagen Reference Standard supplied with the kit.

A list of suggested sample types can be found under the ‘Assay Specification‘ tab.

Assay range

2 – 200µg/ml or 0.25 – 20µg/ml (using optional Collagen Concentration Protocol)

Limit of Detection

2 µg/ml

Detection Method

Colorimetric Detection (556nm) (Endpoint), Requires a microplate centrifuge.

Measurements per kit

96 in total (allows a maximum of 41 samples to be run in duplicate alongside a standard curve).

Suitable Samples

Soluble* collagens from mammalian**:

In-vivo: Tissues, cartilages and fluids.

‍In-vitro: Extracellular matrices / Conditioned media from 2D/3D culture environments.

The straightforward sample processing and analysis of Sirco 2.0 make it a good alternative to conventional hydroxyproline analysis.

‍*Prior salt/acid/acid-pepsin extraction may be necessary to release soluble collagen.

**Sircol 2.0 is primarily designed for use with in-vivo / in-vitro samples of mammalian origin. Collagens originating from other taxonomic groups and kingdoms can also be analysed. See note on p6 of manual for further information.‍

Precautions

This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a microplate centrifuge* (see note below), as well as a spectrophotometer/colorimeter capable of absorbance detection at 556nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.

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*As a minimum, we recommend that the centrifuge can centrifuge a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.

Sircol 2.0 kit contents:

1. Dye Reagent (1x20ml)

2. Collagen Reference Standard (1x5ml, 200µg/ml of soluble Bovine collagen)

3. Plate Wash Reagent (1x28ml)

4. Collagen Concentration Reagent (1x25ml)

5. Neutralisation Reagent (1x8ml)

6. Dye Release Reagent (1x25ml)

7. Assay Microplate (1×96-wells)

8. Microplate Seals (6x)

9. Documentation (QuickStart Guide / Manual / Certificate of Analysis)

NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details. This kit requires the use of a microplate centrifuge, capable of centrifuging a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.