Total Collagen Assay Kit (Perchlorate-Free) (ab222942)

Overview

  • Product name

    Total Collagen Assay Kit (Perchlorate-Free)
    See all Collagen kits
  • Detection method

    Colorimetric
  • Sample type

    Urine, Serum, Other biological fluids, Tissue Homogenate
  • Assay type

    Quantitative
  • Range

    0.5 µg - 10 µg
  • Assay time

    3h 00m
  • Species reactivity

    Reacts with: Other species, Mammals
  • 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 acid 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 acidic 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

  • 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.

  • Typical hydrolyzed Collagen I standard calibration curve.

  • Correlation between collagen standard curve absorbance values obtained using a classical perchlorate-based assay kit and perchlorate-free Total Collagen Assay Kit (Colorimetric) (ab222942).

Protocols

References

ab222942 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Abreviews
This kit in the past has been used to measure total collagen in tissue, serum, and urine however, this kit as well as many other on the market are not tested for cell culture media. Production of Type 1 collagen is a common marker of cells under going differentiation due to an epithelial to mesenchymal (EMT) type process. Here we treated primary fetal membrane cells with oxidative stress inducer (cigarette smoke extract CSE) and measured the amount of total collagen they secreted in 48 hours. To our surprise we were able to detect low levels of collagen from cell media with in the kit range of 0.1ug/ul - 1ug/ul.
To achieve this we used the protocol for urine samples, including adding 4 mg of activated charcoal to each sample to remove the color. However, we did have to individually test each sample to make sure it was neutral. Researchers running this assay on cell media in the future should beware that adding 10N NAOH before hydrolysis and then 10N of HCL after as stated in the book did not neutralize the cells media samples.
Overall, this kit is able to detect low levels of collagen in cell media.

Abcam user community

Verified customer

Submitted Feb 27 2018

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