Soluble Collagen Assay Kit

Catalog No. ABIN5067561

Product Details

Application: Biochemical Assay (BCA)

Characteristics: Soluble Collagen Assay Kit provides a convenient colorimetric method for the detection of soluble collagen from cell or tissue samples. First, the unknown samples or collagen standards are added to a 96 well plate and dried down overnight. Then, a Sirius Red reagent is added to stain the [Gly-x-y] triple helix structure of collagen. Finally, the stained collagen is washed with an Acidic Reagent, eluted from the plate with a Basic Reagent, transferred to a new 96 well plate and measured by a plate spectrophotometer. The amount of collagen in the unknown samples is determined by comparing with a predetermined collagen standard curve. The provided reagents are sufficient for the evaluation of 96 assays including standards and unknown samples.

Components:

1. Collagen Standard : One 500 μL vial containing 3 mg/mL Type I Collagen.
2. Sirius Red Reagent : One 15 mL bottle.
3. Extraction Solution : One 15 mL bottle.
4. 10X PBS : One 10 mL bottle. 2

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Detects soluble collagen in cell and tissue lysates
• Detection sensitivity of approximately 15 ug/ml collagen
• Collagen standard curve included

Reagent Preparation:

1X Acidic Reagent: Add 20 mL of 5X Acidic Reagent to 80 mL of distilled water. Store at 4 °C. Preparation of Standard Curve Prepare a dilution series of Collagen standards in the concentration range of 0 to 500 μg/mL by diluting the Collagen Standard in cold PBS (Table 1). 3 mg/mL Collagen Collagen Standard Tubes Standard (μL) Cold PBS (μL) (μg/mL) 1 100 500 500 2 250 of Tube #1 250 250 3 250 of Tube #2 250 125 4 250 of Tube #3 250 62.5 5 250 of Tube #4 250 31.2 3 6 250 of Tube #5 250 15.6 7 250 of Tube #6 250 7.8 8 0 250 0 Table 1. Preparation of Collagen Standards.

Sample Preparation:

The following recommendations are only guidelines and may be altered to optimize or complement the user's experimental design. 7Cells: Resuspend 1-2 x 10 cells in 1 mL of 0.5 M Acetic Acid containing 0.1 mg/mL Pepsin according to ref. 6 (see p. 7). Disrupt cells on ice by dounce homogenization. Sonicate the homogenate on ice with a probe sonicator. Centrifuge the homogenate at 12,000 x g for 10 minutes. Recover the supernatant and transfer to a new tube. Determine the protein concentration by protein assay. Neutralize the pH of the sample in two steps. First add the Neutralization solution 1:6 directly into the sample. Then add 10X PBS 1:10 directly to the sample to a final concentration of 1X PBS (for example, add 100 μL of Neutralization solution to 500 μL of sample, and then add 67 μL of 10X PBS to the sample). Store unused final sample at -80 °C. Tissue: Homogenize 100 mg of tissue in 1 mL of 0.5 M Acetic Acid containing 0.1 mg/mL Pepsin according to ref. 6 (see p. 7). Disrupt tissue on ice by dounce homogenization. Sonicate the homogenate on ice with a probe sonicator. Centrifuge the homogenate at 12,000 x g for 10 minutes. Recover the supernatant and transfer to a new tube. Determine the protein concentration by protein assay. Neutralize the pH of the sample in two steps. First add the Neutralization solution 1:6 directly into the sample. Then add 10X PBS 1:10 directly to the sample to a final concentration of 1X PBS (for example, add 100 μL of Neutralization solution to 500 μL of sample, and then add 67 μL of 10X PBS to the sample). Store unused final sample at -80 °C. Note: This kit is not recommended for serum, plasma, or urine samples. Perform acid hydrolysis and measure the collagen marker hydroxyproline using Cell Biolabs' Hydroxyproline Assay Kit (STA-675).

Assay Procedure:

1. Prepare and mix all reagents thoroughly before use. Each sample, unknown and standard should be assayed in duplicate.
2. Add 100 μL of collagen standards or unknown samples to a 96 well microplate.
3. Evaporate to dryness overnight in a 37 °C oven for 16 hours.
4. Wash the wells 3 times with 200 μL of distilled water with aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess water. 4
5. Add 150 μL of the Sirius Red Reagent to each well.
6. Incubate for 60 minutes at room temperature on an orbital shaker.
7. Wash the wells 4 times with 200 μL of Acidic Reagent with aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Acidic Reagent.
8. Add 150 μL of Basic Reagent to each well.
9. Incubate 30 minutes at room temperature on an orbital shaker.
10. Transfer 100 μL to the wells of a new plate.
11. Read absorbance of each well on a microplate reader using 540-560 nm as the primary wavelength. 5

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Upon receipt, store the Sirius Red Reagent at room temperature and store the rest of the kit at 4°C. Preparation of Standard Curve Prepare a dilution series of Collagen standards in the concentration range of 0 to 500 μg/mL by diluting the Collagen Standard in cold PBS (Table 1). 3 mg/mL Collagen Collagen Standard Tubes Standard (μL) Cold PBS (μL) (μg/mL) 1 100 500 500 2 250 of Tube #1 250 250 3 250 of Tube #2 250 125 4 250 of Tube #3 250 62.5 5 250 of Tube #4 250 31.2 6 250 of Tube #5 250 15.6 7 250 of Tube #6 250 7.8 8 0 250 0 Table 1. Preparation of Collagen Standards.

Target Details

Background: Collagen serves as the major structural component of animal connective tissues. Collagen is found in fibrous tissues such as skin and ligaments, but is also found in large amounts in bone, cartilage, and cornea. When hydrolyzed, collagen forms gelatin which is used in the food industry as well as medically to treat bone and skin disorders. Collagen makes up about 30 % of the total protein content in animals, making it the most abundant animal protein. Hydroxyproline, a modified form of proline, is found almost exclusively in the protein collagen, in the Y position of the repeating tripeptide Gly-X- Y. By allowing sharp twisting of the collagen helix, hydroxyproline helps to stabilize the structure of collagen. Collagen plays an important structural role in the heart. The cardiac extracellular matrix, which is made up mostly of fibrillar collagen, preserves myocardial integrity, allows for heart force transmission, and contributes to myocyte orientation. Any interruption of the finely balanced regulation of collagen synthesis, post-synthetic assembly, post-translational modification and degradation may have large effects on myocardial function. In addition, collagen has several clinical uses. Collagen has been used extensively in cosmetic surgery to aid in the healing process for burn patients. Collagen has also been used to reconstruct bone in dental, orthopedic, and other surgical scenarios. Collagen is used for bone grafts because of its triple helical structure which makes it a very strong molecule. It is also optimal for use in bones because it does not compromise the structural integrity of the skeleton. Finally, both human and bovine collagen has been used as dermal fillers to treat wrinkling and skin aging.