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Sircol 2.0 Collagen Assay Kit (Soluble)
Soluble collagen

Sircol 2.0 Collagen Assay Kit (Soluble)

Sircol 2.0 is an easy-to-use, dye-based collagen assay for measuring soluble collagen from in-vivo and in-vitro samples. It offers improved sensitivity and a 10-fold reduction in interference from non-collagenous proteins compared with previous Sircol assays, while also being a user-friendly alternative to hydroxyproline analysis.

Product Codes

SIRC2 (96 assays)

SIRC2-Multi (384 assays, MultiPack)

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Key factsHow it worksSuitable samplesBefore you startWhat's in the kitBackgroundFAQ
Research use
Documents
Recently published research
See the papers ↓

Endometrial regeneration

Muscle and tendon remodelling

Bone regeneration scaffolds

At a glance

Key Facts

Assay Range
2–200 µg/ml
0.25–20 µg/ml with the optional Collagen Concentration Protocol
Limit of Detection
2 µg/ml (0.25 µg/ml with the optional concentration protocol)
Detection Method
Colorimetric, 556 nm
Endpoint read; requires a microplate centrifuge
Measurements per kit
96
Up to 41 samples in duplicate, plus a standard curve
Sample Types
In-vivo & in-vitro
Tissues, cartilage, fluids, extracellular matrices and conditioned media
Format
96-well microplate

The kit

Biocolor Sircol 2.0 soluble collagen assay box
Biocolor Sircol 2.0 soluble collagen assay contents
Biocolor Sircol 2.0 assay in use
Biocolor Sircol 2.0 soluble collagen standard curve

Method

How It Works

1
Prepare the sample

Soluble collagen is extracted from tissues, cartilage, fluids, extracellular matrices or conditioned media. Prior salt/acid/acid-pepsin extraction may be necessary to release soluble collagen.

2
Bind the dye

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.

3
Wash and release

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

4
Quantify

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.

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 Sircol 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.‍

Before you start

‍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.

‍*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.

‍

This kit is designed for research use only. Not for use in diagnostic procedures.
‍

Background

About this assay

What is soluble collagen?

Newly made collagen is biochemically active: it plays a part in cell growth, proliferation and differentiation. Because it is still immature, it has minimal cross-linking between collagen molecules. As a result, it can be extracted in soluble form from tissues and cells by cold acid or acid-pepsin digestion, while in fluids and culture media it is already in solution. This fraction is termed soluble collagen, and Sircol 2.0 is designed to measure it.

As collagen matures, it becomes extensively cross-linked and no longer dissolves under these conditions. This is insoluble collagen. To measure it, use our Sircol Insoluble kit.

What's in the kit

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 (1x96-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.

Background

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.

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 the dye-binding chemistry works

How does Sircol 2.0 measure 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 gives a 10-fold reduction in interference from non-collagenous proteins, compared with previous Sircol assays.

Support

Frequently asked questions

The questions our technical team is asked most about this kit. Not answered here?

Ask us directly

How long does the Sircol 2.0 assay take to run?

The total assay time (assuming samples are ready to analyse) is approximately 4-hours, allowing analysis to be completed inside one working day.

What forms of collagen does the Sircol assay measure?

Acid-soluble and pepsin-soluble collagens.

What types of collagen can the Sircol assay be used with?

Mammalian collagens, Types I to V.

How do I prepare samples for measurement with the Sircol 2.0 assay?

Sircol 2.0 measures collagen in solution. Liquids such as culture medium can be used with minimal preparation. Tissues and cell layers first need an extraction step to release their soluble collagen.

  • Culture medium: collect the medium, centrifuge at 1,500 x g for 5 minutes to remove any cellular debris and assay the clear supernatant. Unlike previous Sircol kits, serum-containing medium needs no special pre-treatment.
  • Tissues (e.g. lung, liver, skin, tendon, cartilage, heart valves): rinse away blood, trim off fat and dice into 2–3 mm pieces. Extract overnight (18–24 hours) at 4 °C with shaking in acid-pepsin (0.1 mg/ml pepsin in 0.5 M acetic acid), using 200 µl acid-pepsin solution for every 10 mg of tissue. Centrifuge at 3,000 x g for 10 minutes and assay the supernatant.
  • 2D cell layers and 3D cultures: digest the cells in acid-pepsin overnight at 4 °C, either in the well or after scraping, then clarify at 1,500 x g for 5 minutes. With collagen-based scaffolds, include a cell-free scaffold as a control.

Samples should be free of particles. Neutralise any sample below pH 2.5 or above pH 10. Extracts can be frozen at −20 °C until you're ready to assay.

If your collagen levels are low (below about 20 µg/ml), the optional Collagen Concentration Protocol can be used to pre-process samples and lower the limit of detection from 2 µg/ml to 0.25 µg/ml (it uses a reagent supplied with the kit).

Acid-pepsin extraction releases the soluble collagen fraction. Mature, cross-linked collagen left in the residue can be measured with the Sircol Insoluble kit.

Full protocols are in Part D of the Sircol 2.0 manual, and the concentration protocol is in Part B. You can download it from the Documentation links at the top of this page.

What sort of collagen standard should I use?

For mammalian samples, use the Collagen Reference Standard supplied with the kit (bovine collagen, 200 µg/ml). Sircol 2.0 is calibrated for mammalian collagens, and this standard suits samples from all mammalian species.

Collagens from other groups, such as fish, birds or invertebrates, can also be measured. However, they generally bind less Sircol dye than mammalian collagen. Measured against the bovine standard they would be underestimated, and the limit of detection will be higher.

For accurate results, replace the kit standard with a purified collagen from the same species. Prepare it in the same solvent as your samples and dilute it over the same range as the kit standard (0–200 µg/ml).

The assay measures soluble collagens with an intact triple helix and does not distinguish between collagen types.

What absorbance values can samples be measured at?

Absorbance spectrum maximum for Sircol 2.0 dye in alkali reagent is 556 nm, however absorbance settings within the range 530-560 nm will give suitable readings.

In the literature

How researchers use Sircol 2.0

The newest Sircol, designed for microplate work. Early users are measuring collagen secreted into culture medium and characterising decellularised tissue.

Culture medium

Secreted collagen, with the optional concentration step.

Decellularised tissue

Collagen retained after processing.

Bone & dental biomaterials

Collagen in dentin, bone matrix and graft scaffolds.

Typical samples

Conditioned medium · decellularised tissue · dentin & bone matrix · muscle & tendon

Often used with

Sircol Insoluble
Blyscan™
Fastin™

Selected papers

A curated selection of peer-reviewed work using this kit, chosen to span different tissues, models and questions.

Human Endometrial Extracellular Matrix Hydrogel Facilitated Endometrial Mesenchymal Stem Cells for Endometrial Regeneration

Soluble collagen in native vs decellularised human endometrium. Sircol 2.0 was used on pepsin–acetic acid digests of native and decellularised full-thickness human endometrium, alongside a hydroxyproline assay for total collagen, to confirm collagen was preserved before the matrix was made into EndoGel, a hydrogel for delivering endometrial stem cells in a mouse injury model.

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Advanced Healthcare Materials
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DOI

Caloric restriction and aerobic exercise differentially counteract muscle–tendon dysfunction following diet-induced obesity in C57BL/6J mice

Soluble collagen in muscle and tendon. Sircol 2.0 was used on homogenates of frozen soleus and EDL muscle and tendon tissue from mice, alongside insoluble collagen and elastin, to test how caloric restriction and aerobic exercise counteract muscle–tendon matrix remodelling after diet-induced obesity.

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The Journal of Physiology
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DOI

Cryo-printed collagen scaffolds reinforced with dentin-derived bioactive particles promote osteo-angiogenic bone regeneration

Collagen content of demineralised dentin matrix. Sircol 2.0 was used to compare collagen content between native dentin and processed dentin-derived particles — lyophilised, ground and assayed alongside DNA and sulfated GAG — to confirm matrix composition before the particles were used to reinforce cryo-printed collagen scaffolds for bone regeneration.

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Materials Today Bio
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DOI

Decellularized Aged Bruch's Membrane Confers Unique Biochemical Cues to Retinal Pigment Epithelium for In Vitro Modeling of Age-Related Macular Degeneration

Soluble collagen in native vs decellularised Bruch's membrane. Sircol 2.0 was used on native and decellularised Bruch's membrane from young and aged human donors, characterising an aged matrix scaffold for in-vitro modelling of age-related macular degeneration.

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Aging Cell
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DOI

Evaluating inhalation risks and toxicological impacts of lithium-ion battery thermal runaway emissions

Collagen release after airborne particulate exposure. Sircol 2.0 was used on exposure media from human small airway epithelial cells after 24 hours and 7 days of treatment with thermal runaway particulate matter from LFP and NMC lithium-ion batteries, measuring soluble collagen as a fibrotic marker in an inhalation risk assessment.

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Environment International
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DOI

Binary fabrication of decellularized lung extracellular matrix hybridgels for in vitro chronic obstructive pulmonary disease modeling

Soluble collagen in decellularised lung matrix. Soluble collagen was measured in native lung (~2 mg) and decellularised ECM working material (~15 mg), run immediately before the insoluble assay on the same samples, to characterise hybridgels built for in vitro COPD modelling.

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Acta Biomaterialia
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DOI
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