Sirius Red Total Collagen Detection Assay PLATE Kit

Sirius Red Total Collagen Detection Assay PLATE Kit - Cat Number: 9062P From Chondrex.

Research Field: ECM

Clonality: N/A

Cross-Reactivity:

Host Origin: N/A

Applications: N/A

Isotype: N/A

Detection Range: 500 µg/ml-8 µg/ml

Sample Type: Culture Media, Solubilized Collagen

Concentration: N/A

Immunogen:

PRODUCT SPECIFICATIONS

DESCRIPTION: Assay kit to quantify total collagen levels
FORMAT: 96-well ELISA plate with removeable strips
ASSAY TYPE: Colorimetric assay
ASSAY TIME: < 1 hour
STANDARD RANGE: 500 - 8 µg/ml
NUMBER OF SAMPLES: Up to 80 (duplicate) samples/kit
SAMPLE TYPES: Solubilized tissue, cell culture media, and cultured cells
RECOMMENDED SAMPLE DILUTIONS: Varies depending on sample type
CHROMOGEN: N/A
STORAGE: -20°C
VALIDATION DATA: N/A
NOTES: This product is NOT the Sirius Red Total Collagen Detection Assay Kit (Cat # 9062)

INTRODUCTION

Sirius Red is a unique dye which specifically binds to the [Gly-X-Y]n helical structure on fibrillar collagen (type I to V) and does not discriminate between collagen species and types. Therefore, Chondrex, Inc. provides a Sirius Red Collagen Detection Assay Plate Kit designed to detect the total collagen content in various samples. This kit uses an ELISA plate for various collagen-containing samples such as tissue specimens, cell culture media, and cultured cells. This protocol is ideal for users who have many samples because the total assay working time is less than 60 minutes and 80 samples can be measured in duplicate. Due to the low level of collagen in cell culture media, additional concentration steps may be necessary. Concentrating Solution (Cat # 90626) for cell culture media samples is NOT included in this kit. Please contact customer service support@chondrex.com to place an order.
For determining levels of collagen from individual species or different types of samples, Chondrex, Inc. recommends the Sirius Red Collagen Detection Assay Kit using the tube assay method (Cat # 9062), Type I Collagen Detection ELISA Kits, and the Type II Collagen Detection ELISA Kit. Please visit www.chondrex.com for more information. For convenience, the same sample preparation methods may be used for both the Collagen Detection ELISA Kits and the Sirius Red Collagen Detection Assay Kits.

PREPARING SAMPLES

Tissue specimens and cultured cells can be used, however solid samples must be solubilized for this assay. Culture media samples may
require a concentrating process. In addition, heat-denatured collagen tends to have a lower binding affinity for Sirius Red, resulting in
underestimated values. Chondrex, Inc. provides “Tips for Collagen Solubilization” to prepare pepsin-soluble collagen samples. Please
contact customer service support@chondrex.com for more information. Depending on the solubilization method, these soluble collagen
samples can be used:
1. Salt soluble collagen (0.15M NaCl in 0.1M Tris-HCl, pH 7.4)
2. Acid soluble collagen (0.05M acetic acid)
3. Pepsin soluble collagen (0.05M acetic acid with pepsin)

CULTURE MEDIA

Samples containing higher concentrations of serum can cause high background values. Therefore, Chondrex, Inc. recommends reducing
the serum supplement concentration in samples such as cell culture media to lower than 5% using 0.05M PBS pH 7.4.
SAMPLE CONCENTRATION
The concentration of collagen in culture media is generally low, making it difficult to detect collagen within the standard range of this kit.
Chondrex, Inc. recommends a sample concentration process using Concentrating Solution (Cat # 90626). Furthermore, a negative control
using culture media should be used as this concentration method may result in elevated background levels.
1. Take 1 ml culture medium.
2. Add 250 l of Concentrating Solution.
3. Vortex and incubate at 4°C for 16-24 hours.
4. Centrifuge at 10,000 rpm for 3 minutes.
5. Discard supernatant.
6. Add 100 l of 0.05M acetic acid to dissolve the pellet. Use this solution as sample.
7. Calculated collagen concentration should be multiplied by a 0.1 dilution factor.

NOTES BEFORE USING ASSAY
NOTE 1: It is recommended that the standard and samples be run in duplicate.
NOTE 2: Warm up all buffers to room temperature before use.
NOTE 3: Measure exact volume of buffers using a serological pipet, as extra buffer is provided.
NOTE 4: Partially used stock reagents may be kept in their original vials and stored at -20⁰C for use in a future assay.