1Sirius Red Total Collagen Detection Assay Plate Kit
Catalog # 9062P

For Research Use Only - Not Human or Therapeutic Use

PRODUCT SPECIFICATIONSDESCRIPTION:

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/ANOTES:This product is NOT the Sirius Red Total Collagen Detection Assay Kit (Cat # 9062)© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

2Sirius Red Total Collagen Detection Assay Plate Kit
Catalog # 9062P

For Research Use Only - Not Human or Therapeutic Use

INTRODUCTIONSirius 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 lessthan 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 CollagenDetection 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 forboth the Collagen Detection ELISA Kits and the Sirius Red Collagen Detection Assay Kits.

KIT COMPONENTS
Item

Quantity

AmountStorageStandard - Bovine Type I Collagen (90621)
Sirius Red Solution (90622)
Washing Solution (90623)

1 vial
1 bottle
1 bottle

0.5 mg/ml, 1 ml
50 ml
50 ml

-20°C
-20°C
-20°C/*

Extraction Solution (90624)
1 bottle
30 ml
0.5M Acetic Acid (10X Acetic Acid) (90625)
1 bottle
20 ml
Flat-bottom 96-Well ELISA Plate (9026)
2 each8-well strips x 12
V-bottom 96-Well Plate for Centrifugation (90261)
2 each2 plates
Flexible 96-Well Plate for Sample Dilution (90262)
1 each1 plate96-Well ELISA Plate Sealer (90263)
2 each2 each*Washing solution, extraction solution, 0.5M acetic acid, and plates can also be stored at room temperature.

-20°C/*-20°C/*-20°C/*-20°C/*-20°C/*-20°C/*© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

3PREPARING 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 inunderestimated values. Chondrex, Inc. provides “Tips for Collagen Solubilization” to prepare pepsin-soluble collagen samples. Pleasecontact customer service support@chondrex.com for more information. Depending on the solubilization method, these soluble collagensamples 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 CONCENTRATIONThe 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.
NOTE 5: This kit contains animal components from non-infectious animals and should be treated as potential biohazards in use and fordisposal.

© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

4

PLATE LAYOUT
123

4

5

678

9101112A

500

500

S1S1S9S9S17

S17S25

S25

S33

S33

B250

250

S2S2S10S10

S18

S18

S26

S26

S34

S34C125125

S3S3S11

S11

S19

S19

S27

S27

S35

S35

D6363

S4S4S12S12S20

S20

S28

S28

S36

S36

E3131

S5S5

S13

S13

S21

S21

S29

S29

S37

S37

F1616

S6

S6

S14

S14

S22

S22

S30

S30

S38

S38

G88

S7

S7

S15

S15

S23

S23

S31

S31

S39

S39

HB

B

S8

S8

S16

S16

S24

S24

S32

S32

S40

S40

Standards

SamplesFigure 1 - Removing Supernatant by Pipetting

ASSAY OUTLINE
Add 50 l of diluted standards and samples to wells of V-bottom 96-well plate

Add 250 l of Sirius Red Solution to wells of V-bottom 96-well plateMix and incubate at room temperature for 20 minutes.
Centrifuge at 3,000 rpm for 10 minutes
Carefully aspirate 250 l of supernatant and discard.
Add 250 l of Washing Solution to wells of V-bottom 96-well plateMix and centrifuge at 3,000 rpm for 10 minutes
Carefully aspirate 250 l of supernatant and discard.
.
Add 150 l of Extraction Buffer to wells of V-bottom 96-well plate

Carefully pipet to dissolve pellets completely

Transfer 150 l from each well to a new flat-bottom 96-well plate

Read plates at 530 nm (510-550 nm)© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

5ASSAY PROCEDURE
1.

Prepare 1X Acetic Acid: using purified distilled water, prepare a 1X acetic acid (0.05M) solution for the standards and samples.

2.

Prepare Standard Solutions: using a flexible 96-well plate, centrifuge tubes, or disposable culture tubes, add 125 µl of 0.05M acetic
acid to seven blank spaces. Mix 125 µl of Standard (500 µg/ml) with an equal amount of 0.05M acetic acid (250 µg/ml). Repeat thisprocedure five times to make 125, 63, 31.5, 16, and 8 µg/ml standard solutions.
125 l1000 l125 l125 l125 l125 l125 l125 l125 l125 l125 l125 l125 l125 l500 g/ml

250 g/ml

125 g/ml

63 g/ml

31.5 g/ml

16 g/ml

8 g/ml

Blank

①②③④⑤

⑥

⑦

⑧

3.

Prepare Sample Solutions: prepare samples using flexible 96-well plates, small centrifuge tubes, or disposable culture tubes. Because
the collagen concentration of samples is unknown, preparing multiple samples with various dilutions using 0.05M acetic acid isrecommended.

4.

Add Standards and Samples: add 50 µl of blank, diluted standard solutions, and samples to a V-bottom 96-well plate in duplicate.

5.

Add Sirius Red Solution: add 250 µl of Sirius Red Solution to each well. Pipet several times to mix the standards and samples withthe dye solution and then apply the plate sealer. Incubate for 20 minutes at room temperature.

6.

Centrifuge: centrifuge at 3,000 rpm for 10 minutes.

7.

Discard Supernatant: remove the plate sealer, tilt the plate at a 45-degree angle (Figure 1), and aspirate 250 µl of supernatant without
disturbing the pellet. If the pellet is disturbed, centrifuge again before removing the supernatant. Discard supernatant.

NOTE: Figure 2 shows typical pellets in the standard wells after removing supernatants

Figure 2 - Typical Pellets in the Wells

Figure 3 - Typical Pellets in the Wells

Figure 4 - Typical Color in the Wells

8.

Add Washing Solution: add 250 µl of Washing Solution to each well and re-apply the plate sealer.

9.

Centrifuge: centrifuge at 3,000 rpm for 10 minutes.

© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.0
16928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com

610. Discard Supernatant: remove the plate sealer, tilt the plate at a 45-degree angle (Figure 1), and aspirate 250 µl of supernatant without
disturbing the pellet. If the pellet is disturbed, centrifuge again before removing the supernatant. Discard supernatant.
NOTE: Figure 3 shows typical pellets in the standard wells after removing supernatant.
11. Add Extraction Buffer: add 150 µl of Extraction Buffer to each well. Pipet several times to dissolve the pellet completely.
NOTE: Figure 4 shows typical color in the standard wells after dissolving the pellet.
12. Transfer: transfer 150 µl from each well to a clean, flat-bottom 96-well plate.
13. Read Plate: read the plate at 530 nm (510-550 nm).

CALCULATING RESULTS
1.

Average the duplicate OD values for the blank, standards, and test samples.

2.

Subtract the “blank” (B) values from the averaged OD values in step 1.

3.

Plot the OD values of standards against the concentration of the standard (µg/ml). Using a log/log plot will linearize the data. Figure 5
shows a representative experiment where the standard range is 8 - 500 µg/ml.

4.

The µg/ml of collagen in test samples can be calculated using regression analysis. Multiply by the sample dilution factor to obtain thecollagen concentration in the original sample specimens. If the OD values of the samples are outside of the standard curve range, there-assay the samples at different dilutions.

Figure 5 - A Typical Standard Curve for the Sirius Red Total Collagen Detection Assay Plate Kit
10.00
R² = 0.9982

OD 530 nm

1.00

0.10

0.01
110100Collagen Concentration (g/ml)

1000

TROUBLESHOOTING
For frequently asked questions about assays and ELISAs, please see Chondrex, Inc.’s Assay FAQ for more information.

© 2020 Chondrex, Inc. All Rights Reserved, 9062P 2.016928 Woodinville-Redmond Rd NE Suite B-101
Woodinville, WA 98072

Phone: 425.702.6365 or 888.246.6373
Fax: 425.882.3094

www.chondrex.com
support@chondrex.com