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. Chose 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 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 4ºC due to the unwinding of the triple helix and formation of random chains.

‍Overview of the Sircol assay process:

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

Limit of Detection

100µg/ml

Detection Method

Colorimetric Detection (556nm) (Endpoint)

Measurements per kit

110 in total (allows a maximum of 46 samples to be run in duplicate alongside a standard curve).

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 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.

Precautions

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.

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.

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.

Introduction

HiPure DNA Clean Up Kit usesproprietary chemistry and HiPure technology to recover DNA Fragments between20bp-20kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing,restriction digestion, or various labeling reactions. In addition, this kit canbe also used to recover DNA directly from enzymatic reactions such as PCR andenzyme digestion reactions.

Details

Specifications

Features	Specifications
Main Functions	Recover DNA fragments between 20bp-20kbp from crude DNA and enzymatic reaction solution
Applications	PCR, sequencing, labeling reactions, ligations and restriction digestion, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Crude DNA, enzymatic reaction solution
Sample amount	<150μl
Recovery	80%
Elution volume	≥15μl
Time per run	≤10 minutes
Liquid carrying volume per column	800µl
Binding yield of column	20µg

Principle

The HiPure system uses a simple bind-wash-elute procedure. Gelslices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the column. Nucleic acids adsorb to the silica-gel membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in subsequent applications.

Advantages

• High recovery efficiency – up to 80% DNA recovery
• Fast – isolation can be completed in 10 minutes by column gel method
• Low elution volume – elution volume can be as low as 10μl
• Efficient – efficient removal of various inhibitory factors

Kit Contents

Contents	D214102	D214103
Purification Times	100 Preps	250 Preps
Buffer GWP	30 ml	60 ml
Buffer DW2	20 ml	50 ml
Elution Buffer	30 ml	60 ml
HiPure DNA Mini Columns I	50	250
2 ml Collection Tubes	50	250

Storage and Stability

HiPure DNA Clean Up Kit components are guaranteed for at least one year when stored at room temperature. If any precipitates form in the buffers, warm at 37℃ to dissolve.

HiPure DNA Clean Up Kit usesproprietary chemistry and HiPure technology to recover DNA Fragments between20bp-20kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing,restriction digestion, or various labeling reactions. In addition, this kit canbe also used to recover DNA directly from enzymatic reactions such as PCR andenzyme digestion reactions.

Description

The EzRNA™ T7 High Yield RNA Synthesis Kit is a user-friendly product for enzymatic RNA production. The enzyme mix contains adequate amount of T7 RNA polymerase, pyrophosphatase, and RNase inhibitors for in vitro transcription (IVT). Along with 10X Transcription Buffer and NTP Premix, users can swiftly assemble IVT reactions without compromising RNA yield. The EzRNA™ T7 High Yield RNA Synthesis Kit allows for the attainment of approximately up to 150 µg RNA yield within 2 hours at 37°C.

Features

• High yield
• Versatile- suitable for short and long transcripts
• NTP premixed- Minimal pipetting and setup time
• Compatible with CleanCap® Reagent AG
• Lithium chloride included for RNA purification

Application

• Generation of RNA from T7 promoter-driven DNA sequences
• Suitable for subsequent cap-0 and cap-1 modification

Storage

-20°C for 12 months

The EzRNA™ T7 High Yield RNA Synthesis Kit is a user-friendly product for enzymatic RNA production. The enzyme mix contains adequate amount of T7 RNA polymerase, pyrophosphatase, and RNase inhibitors for in vitro transcription (IVT). Along with 10X Transcription Buffer and NTP Premix, users can swiftly assemble IVT reactions without compromising RNA yield. The EzRNA™ T7 High Yield RNA Synthesis Kit allows for the attainment of approximately up to 150 µg RNA yield within 2 hours at 37°C.