Total Collagen Assay Kit (Perchlorate-Free) (ab222942)
Key features and details
- Assay type: Quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 3 hr
- Sample type: Serum, Tissue Homogenate, Urine
Overview
-
Product name
Total Collagen Assay Kit (Perchlorate-Free)
See all Collagen kits -
Detection method
Colorimetric -
Sample type
Urine, Serum, Tissue Homogenate -
Assay type
Quantitative -
Range
0.5 µg - 10 µg -
Assay time
3h 00m -
Species reactivity
Reacts with: Mammals, Other species -
Product overview
Total Collagen Assay Kit (Perchlorate-Free) (ab222942) provides a quick and convenient method to detect collagen in tissue lysates as well as biological fluids such as urine or serum.
The collagen assay protocol is based on the Alkaline hydrolysis of samples to yield free Hydroxyproline. The released hydroxyproline is oxidized to form a reaction intermediate, which further in reaction forms brightly-colored chromophore that can be easily detected at OD 560 nm.
The assay employs a proprietary developer solution to accurately measure collagen in hydrolysates without the use of hazardous perchloric acid, removing the need for any special handling and waste-disposal protocols. The assay can detect as low as of 0.5 µg collagen/well.
Collagen assay protocol summary:
- hydrolyze samples and standard with concentrated NaOH for 1 hr at 120°C
- cool on ice and neutralize with concentrated HCL
- centrifuge at 10,000g for 5 min to pellet debris
- add samples and standards to wells
- evaporate wells to dryness at 65°C
- add oxidation mix and incubate for 20 min
- add developer and incubate for 5 min
- add DMAB concentrate and incubate for 45 min at 65°C
- analyze absorbance at 560 nm with a microplate reader -
Notes
Collagen is the most abundant insoluble protein found in the extracellular matrix and connective tissues. It can be found in skin, tendons, bone, cartilage, muscle, vitreous humor and ligaments, among other tissues. There are more than sixteen - well characterized types of collagens, but types I, II and III collagen comprise more than 80% content in mammals.
-
Platform
Microplate reader
Properties
-
Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Chloramine T Concentrate 1 x 600µl Collagen I Standard 1 x 200µl Developer 1 x 5ml DMAB Concentrate 1 x 5ml Oxidation Buffer 1 x 10ml Plate sealer 1 unit
Images
-
Total Collagen Assay Kit (Perchlorate-Free) (ab222942)
Estimation of total collagen content in rat tissues. Rat leg muscle, spleen, heart and lung samples were prepared followind the sample preparation procedure described in the protocol (homegenized in ddH2O, hydrolyzed with 10 N NaOH for 1 hour at 120°C and neutralized with 10 N HCl). For each sample, 10 μL of the final neutralized hydrolysate was assayed. Total Collagen levels (calculated as μg collagen/mg wet tissue) for the samples were: 13.80 +- 0.73 ug/mg for muscle, 3.77 +- 0.54 ug/mg for spleen, 4.55 +- 0.81 ug/mg for heart and 12.51 +- 1.27 ug/mg for lung. Data are mean ± SEM of 3-4 replicates.
-
Total Collagen Assay Kit (Perchlorate-Free) (ab222942)Typical hydrolyzed Collagen I standard calibration curve.
-
Total Collagen Assay Kit (Perchlorate-Free) (ab222942)Correlation between collagen standard curve absorbance values obtained using a classical perchlorate-based assay kit and perchlorate-free Total Collagen Assay Kit (Colorimetric) (ab222942).
