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).
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. Chose our Sircol 2.0 collagen kit if you need to analyse SOLUBLE 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.
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 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.
Assay range
100 – 1000 µg/ml
100µg/ml
Colorimetric Detection (556nm) (Endpoint)
110 in total (allows a maximum of 46 samples to be run in duplicate alongside a standard curve).
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 includes tissues, bone and calcified tissue.
*Insoluble collagens must be converted into soluble form prior to assay. Instructions and regents 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.
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.
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.
As collagens mature, they become increasingly crosslinked and insoluble – characteristics necessary for key biophysical role that collagen plays in living organisms. Biocolor’s Sircol™ INSOLUBLE Collagen Kit is a dye-binding assay designed for accurate quantification and measurement such collagens. It is ideal for analyzing crosslinked / insoluble collagens from sources such as tissues, bone, and calcified tissue.
endo-BCN-PEG2-acid is a PEG linker containing a BCN group and a terminal carboxylic acid. The BCN group can react with azide-tagged biomolecules. The terminal carboxylic acid can react with primary amine groups in the presence of activators (e.g. EDC, or HATU) to form a stable amide bond. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
endo-BCN-PEG2-acid is a PEG linker containing a BCN group and a terminal carboxylic acid. The BCN group can react with azide-tagged biomolecules. The terminal carboxylic acid can react with primary amine groups in the presence of activators (e.g. EDC, or HATU) to form a stable amide bond. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
Usages：
Determination of coliform and fecal coliform for multiple tube fermentation.
Principle:
Tryptone provide carbon and nitrogen sources to meet the needs of bacterial growth; sodium chloride osmotic pressure balance can be maintained; Lactose is a coliform fermentable sugars; potassium dihydrogen phosphate and dipotassium phosphate is a buffer; lauryl sodium can inhibit the growth of non-coliform bacteria.
Formulation (per liter):
Trypticase:20g
Sodium chloride: 5g
Lactose:5g
Potassium dihydrogen phosphate: 2.75g
Dipotassium hydrogen phosphate :2.75g
Sodium lauryl sulfate 0.1g
Final pH6.8 ± 0.2
How to use:
1. Suspend 35.6g of the product, adding 1 L of distilled or deionized water, heated to boiling stirring until completely dissolved, packed in a test tube with a small down tube, 121 ℃ autoclave 15min, leave to cool to room temperature, standby .
2.Sample handling and dilution.
3.Selected three consecutive dilution, each dilution was inoculated three LST broth tubes, each tube was inoculated 1mL.
4. Put the tubes in an incubator 36 ± 1 ℃ cultured for 48 ± 2h.
5. Observe the results. if all LST broth don’t pruduse gas, can be reported as negative for E. coli, if gas production then will have to make further confirmed by experiments.
Quality control:
Quality control strains were inoculated and culuture at 36 ± 1 ℃ for 24h ,results are as follows:
Bacterial Name Bacterial No. Growth Status Gassing
Escherichia coli ATCC25922 good +
Salmonella typhimurium CMCC (B) 50115 good —
Staphylococcus aureus ATCC6538 inhibited —
Storage: Store in a dark, cool and dry place, tighten the cap immediately after use. Storage period of three years.
Specifications: 250g/bottle
250g