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Sircol™ Insoluble Collagen assay kit
Insoluble collagen

Sircol™ Insoluble Collagen assay kit

As collagens age, they become increasingly crosslinked and insoluble, characteristics necessary for the structural role they play in tissues such as bone. Biocolor's Sircol™ INSOLUBLE Collagen Kit is an easy-to-use dye-binding assay that can be used to accurately measure the amount of insoluble collagen content in both in-vivo and in-vitro tissues.
Our previous S1000 Sircol Soluble Collagen kit can be found here.
NB: For quantification of soluble/immature collagens we recommend our Sircol 2.0 soluble collagen kit. here.
Our new Sircol 2.0 Soluble Collagen kit can be found here.

Product Codes

S2000 (110 assays)

‍S6000 (440 assays, MultiPack)

UK & Ireland orders are supplied direct from Northern Ireand. For other locations please order through our distributor network.

Order through your local distributorUK & Ireland customers: order direct
Key factsHow it worksSuitable samplesBefore you startWhat's in the kitBackgroundFAQ
Research use
Documents
Recently published research
See the papers ↓

Bioprinted tumour models

Cartilage tissue engineering

Cardiac matrix scaffolds

At a glance

Key Facts

Assay Range
100–1000 µg/ml
Limit of Detection
100 µg/ml
Detection Method
Colorimetric, 556 nm
Endpoint read
Measurements per kit
110
Up to 46 samples in duplicate, plus a standard curve
Sample Types
In-vivo tissue
Tissues, bone and calcified tissue
Format
1.5ml Microtubes

The kit

Biocolor Sircol Insoluble collagen assay kit box
Sircol Insoluble collagen assay kit contents
Sircol dye released from Insoluble collagen in Biocolor's Sircol assay kit
Standard Curve for Sircol Insoluble collagen assay kit

Method

How It Works

1
Fragment the sample

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, which can then be assayed.

2
Bind the dye

Addition of Sircol Dye Reagent to these pre-treated 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.

3
Release and read

The denatured collagen-bound dye is then eluted and measured spectrophotometrically at 556nm.

4
Quantify

The insoluble collagen content of unknown samples is quantified by comparison against a calibration curve prepared using the denatured collagen standard supplied with the kit.

Suitable samples

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. Sources of material include tissues, bone and calcified tissue.

*Insoluble collagens must be converted into soluble form prior to assay. Instructions and reagents are provided with the kit. Depending on 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 species under assay.‍

Many customers have found that the straightforward sample processing and analysis of Sircol make it a good alternative to conventional hydroxyproline analysis.

Before you start

This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a centrifuge, water bath / heated block, 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.

Background

About this assay

What's in the kit

Sircol Insoluble Collagen kit contents:‍

1. Sircol Dye Reagent (1x110ml)

2. Denatured Collagen Reference Standard (1x5ml, 1.0mg/ml)

3. Acid-Salt Wash Reagent (1x20ml)

4. Fragmentation Reagent (1x110ml)

5. Alkali Reagent (1x110ml)

6. 2ml screw-cap tubes for preparation of samples.

7. Assay kit manual

NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details.

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.

This version of the kit is designed to detect and measure INSOLUBLE forms of collagen. Choose our Sircol 2.0 collagen kit if you need to analyse SOLUBLE 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 the dye-binding chemistry works

How does the Sircol assay detect collagen?

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 organisation 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 due to the unwinding of the triple helix and formation of random chains.

Support

Frequently asked questions

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

Ask us directly

What absorbance values can samples be measured at?

Absorbance spectrum maximum for Sircol dye in alkali reagent is 556 nm, however absorbance settings within the range 520-570 nm will give suitable readings.

Why does the Sircol Insoluble collagen assay demonstrate lower absorbance values when compared to the Sircol Soluble collagen assay?

Maximal binding of Sircol dye occurs in collagens possessing intact triple helix organisation since the highly ordered [Gly-X-Y]n helical structure of tropocollagen contributes to dye binding. Affinity is progressively reduced during heat denaturation (>45°C) due to the unwinding of the triple helix and formation of random chains. Since samples and standards for the Sircol Insoluble collagen assay are in a denatured state (necessary to convert insoluble 'solid' samples into the liquid phase for assay) they will therefore bind less dye than the same amount of material in the non-denatured state.

The collagen pellet is much smaller than the pellets in the S1000 Soluble collagen assay, why is this?

Samples in the Sircol Insoluble collagen assay are gently denatured via treatment with Sircol Fragmentation Reagent. This denaturation causes some loss of collagen structure. The dye-bound pellet will therefore be more 'dense' as it is able to pack more tightly following centrifugation.

In the literature

How researchers use Sircol Insoluble

Most often used as the second half of a ‘total-collagen’ measurement. Run Sircol on the soluble fraction, then Sircol Insoluble on what remains, to capture mature, crosslinked collagen.

Total collagen

Soluble plus insoluble on the same sample.

Decellularised scaffolds

Crosslinked collagen retained.

Fibrosis & mature scar

Insoluble collagen in established fibrosis.

Engineered tissue

Collagen matrix laid down by cells.

Typical samples

Decellularised tissue · lung and heart tissue · engineered constructs · tissue residue after soluble extraction

Often used with

Sircol Original
Blyscan™
Fastin™

Selected papers

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

Cryobioprinted human tumor models with shelf-stable programmability

Native collagen in decellularised porcine pancreas. Sircol Insoluble was used on native and decellularised porcine pancreas matrix, confirming collagen was retained after decellularisation, in building cryopreservable pancreatic tumour–stroma bioinks for off-the-shelf drug-screening models.

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Trends in Biotechnology
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DOI

JMJD3 and UTX as key targets for gene-modified mesenchymal stem cell therapy in cartilage tissue engineering

Insoluble collagen in implanted gene-modified stem cell constructs. Sircol Insoluble was used on alginate-bead constructs of JMJD3-, UTX- or GFP-transfected mesenchymal stem cells, implanted subcutaneously in mice for four weeks, to assess cartilage matrix deposition.

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Molecular Therapy
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DOI

Human iPSC-derived cardiac-specific extracellular matrix scaffolds for cardiomyocyte maturation and post-myocardial infarction repair

Insoluble collagen in cell-derived ECM scaffolds. Alongside the soluble fraction, insoluble collagen was quantified in engineered ECM sheets from human iPSC-derived cardiac fibroblasts, primary cardiac fibroblasts and human dermal fibroblasts, giving a measure of the crosslinked matrix in scaffolds used for cardiomyocyte maturation and post-infarction repair.

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Bioactive Materials
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DOI

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

Insoluble collagen in decellularised lung matrix. Insoluble collagen assays were run directly after the soluble assays on the same native and decellularised lung samples, resolving the crosslinked fraction of the matrix used to build COPD hybridgel models.

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Acta Biomaterialia
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DOI
Explore research using all Biocolor kits →

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