Description
The Biocolor Sircol™ 2.0 Soluble Collagen Assay is designed for quantitative measurement of soluble collagen in material from in-vivo and in-vitro sources. The kit employs colorimetric detection in a convenient microplate-based format.
This latest addition to the highly cited family of "Sircol™" collagen kits combines an updated microplate format with an improved dye formulation to significantly enhance collagen sensitivity and specificity. This makes it possible to directly analyze collagen from complex samples such as conditioned media during 2D or 3D cell culture.
NOTE: This assay is not suitable for covalent cross-linked collagen. However, the Sircol Insoluble Collagen Assay (S2000) replaces hydroxyproline analysis. This allows the customer to measure insoluble collagen within one working day.
NOTE: We recommend that new customers use our Sircol 2.0 assay kit as it improves sensitivity, specificity, and user-friendliness; however, we will continue to offer the older, "original" kit (cat. no. S1000) and reagents as well.
Features
- User-Friendly Format: Convenient microplate format allows up to 96 parallel measurements.
- Straightforward Analysis: Uses reliable, dye-based colorimetric detection (556 nm).
- Enhanced Sensitivity: Detect collagen at concentrations starting from 2 μg/ml (0.25 μg/ml with optional concentration protocol, sample permitting).
- Reduced Interference: Experience a *10-fold reduction in interference from non-collagenous proteins (*compared to previous Sircol™ assays)
Specifications
| Assay Range |
|
Optional Collagen Concentration Protocol requires a minimum of 1 ml sample volume. |
| Limit of Detection |
|
In-vitro samples containing added animal serum will exhibit a slightly reduced limit of detection, approx. 2-10 μg/ml, serum depending. |
| Detection Method | Colorimetric dye-binding assay, 556 nm | Wavelengths between 530 – 560 nm are suitable for analysis. |
| Measurements per Kit | 96 in total | Allows a maximum of 41 samples to be run in duplicate alongside a standard curve. |
| Suitable Samples |
Liquid extracts containing soluble* collagen.
|
* Prior salt/acid/acid-pepsin extraction may be necessary to release soluble collagen. |
| Sample Volume | 10 – 100 μl | If using optional Collagen Concentration Protocol, 1000 μl is required. |
| Reactivity |
Soluble collagens containing intact triple-helix structure. As supplied, Sircol™ 2.0 is calibrated for samples of mammalian* origin. |
Sircol™ 2.0 does not distinguish between different types of the collagen family. * See explanation below. |
Principle of Method
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).
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.
Standard Curve (Example)
In-vivo Samples
In addition to in-vitro samples, Sircol 2.0 Assay can be used to measure collagen in tissue samples from in-vivo sources, such as muscle, lung, liver, cartilage, tendons, aorta, valves, skin, etc. These are usually obtained by biopsy or dissection and may be processed as "wet" tissue or freeze-dried for processing as "dry" samples.
The assay manual provides an acid-pepsin extraction protocol along with guidelines on sample collection and preparation.
In-vitro Samples
In addition to in-vivo samples, Sircol 2.0 Assay can be used to measure collagen from in-vitro samples. These are generally obtained following culture of animal cells in either 2D (conventional) or modern 3D cell culture systems.
Suitable sample sources include:
- Conditioned cell culture medium: Many types of cells secrete collagen into the extracellular space when cultured in-vitro. This collagen is naturally soluble and accumulates in the growth medium that bathes the cells. For this reason, conditioned cell culture medium samples can be readily analyzed by Sircol 2.0 Assay.
- Extracellular matrix (ECM): This is deposited onto substrates over (extended) cell culture time. These include:
- T-flasks or microwell plates as used in conventional 2D cell culture.
- 3D culture matrices (provided they are not composed of collagen-derived
materials).
Species
Customers are advised that the Sircol™ 2.0 kit has been calibrated for samples of mammalian origin by virtue of the included mammalian-derived Collagen Reference Standard. Analysis of collagens from other taxonomic groups or kingdoms is possible. However, the published limit of detection will be reduced since non-mammalian collagens generally bind less dye. For accurate quantification customers should replace the kit Collagen Reference Standard with a species-matched, purified preparation of collagen.
Kit Contents & Storage
| Component | Volume Supplied |
Storage Recommendations (once kit is opened) |
| Dye Reagent |
20 ml | Room temperature |
| Collagen Reference Standard (200 μg/ml) |
5 ml |
4°C |
| Plate Wash Reagent |
28 ml |
4°C |
| Collagen Concentration Reagent |
25 ml |
4°C |
| Neutralization Reagent |
8 ml |
Room temperature |
| Dye Release Reagent |
25 ml |
Room temperature |
| Assay Microplate | 1 x 96 wells | Room temperature |
| Microplate Seals | 6x | Room temperature |
| Documentation |
|
N/A |
Resources
Related Products
- Biocolor Sircol™ Soluble Collagen Assay (1.0 version)
- Biocolor Sircol™ Insoluble Collagen Assay
- Collagen and Hydroxyproline Assays
- Extracellular Matrix Assays
- Extracellular Matrix Molecules
Ilex Life Sciences LLC is an official distributor of Biocolor Ltd. products.