• Product name

    Hydroxyproline Assay Kit (Colorimetric)
  • Detection method

  • Sample type

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

  • Range

    0.1 µg - 1 µg
  • Assay time

    3h 00m
  • Species reactivity

    Reacts with: Other species, Mammals
  • Product overview

    Hydroxyproline Assay Kit (Colorimetric) ab222941 provides a quick and convenient method to quantify hydroxyproline in tissue lysates and biological fluids such as urine and serum.

    The classical hydroxyproline assay protocol is based on the oxidation of hydroxyproline to a pyrrole intermediate followed by reaction with Ehrlich’s reagent dissolved in concentrated perchloric acid. Perchloric acid is a hazardous material that is both toxic and highly reactive, requiring special handling and waste-disposal protocols.

    This hydroxyproline assay protocol employs a proprietary acidic developer solution to accurately measure hydroxyproline in hydrolysates without the use of hazardous perchlorates. It is a quick and convenient protocol where hydroxyproline gets 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 can detect as low as 0.05 µg hydroxyproline/well.

  • Notes

    Hydroxyproline assay protocol summary:
    - add 10 N concentrated NaOH to samples and hydrolyze at 120ºC for 1 hr
    - cool on ice
    - neutralize with 10 N concentrated HCl, centrifuge and collect supernatant
    - add samples and standards to wells
    - evaporate wells to dryness by heating at 65ºC
    - add oxidation reagent mix to dissolve crystalline residue, and incubate at room temp for 20 min
    - add developer and incubate at 37ºC for 5 min
    - add DMAB concentrate and incubate for 45 min at 65ºC
    - analyze with microplate reader

  • Platform

    Microplate reader


  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    Chloramine T Concentrate 1 x 600µl
    Developer 1 x 5ml
    DMAB Concentrate 1 x 5ml
    Hyp Standard 1 x 100µl
    Oxidation Buffer 1 x 10ml
    Plate sealer 1 unit
  • Research areas

  • Relevance

    Hydroxyproline, a non-essential amino acid derived from proline, with no known therapeutic use. Hydroxproline is used as a major component of structural protiens such as collagen, connective tissues, plant cell walls, tendons and ligaments and provides skin elasticity. Vitiman C is required for the conversion process from proline to hydroxyproline, a deficincy in vitiman C can lead to defects in collagen synthesis, thus, resulting in easy bruising, internal bleeding, breakdown of connective tissue of the ligaments and tendons, and increased risk to blood vessel damage. An unusual feature of this amino acid is that, it is not incorporated into collagen during biosynthesis at the ribosomal level, but is formed from proline by a posttranslational modification by an enzymatic hydroxylation reaction.


  • Typical Hydroxyproline standard calibration curve.



This product has been referenced in:

  • Yang HY  et al. Dendropanax morbifera Ameliorates Thioacetamide-Induced Hepatic Fibrosis via TGF-ß1/Smads Pathways. Int J Biol Sci 15:800-811 (2019). Read more (PubMed: 30906211) »
  • El-Agamy DS  et al. Pristimerin protects against doxorubicin-induced cardiotoxicity and fibrosis through modulation of Nrf2 and MAPK/NF-kB signaling pathways. Cancer Manag Res 11:47-61 (2019). Read more (PubMed: 30588110) »
See all 2 Publications for this product

Customer reviews and Q&As

Collagen hydrolysate (CH) is a water-soluble substance, which together with the ability of fish to absorb compounds from the water through the gills [1], should allow its penetration. We tested the possibility of administering the drug to fish through water for absorption into the bloodstream. The fish were treated daily for a week with CH compound in three different doses: 10, 20 and 40 mg/L, compared with a control group (without CH). To assess the amount of CH in the serum, blood was extracted and hydroxyproline (HYP) was detected by using Hydroxyproline Assay Kit according to manufacturer’s protocol.
The values obtained were lower than the values of the calibration curve, these results can be explained by two possible reasons, it is possible that the material is not absorbed by the fish. Another possibility is that the values are lower than the identification threshold of the kit.

Rubinstein, A.L., Zebrafish assays for drug toxicity screening. Expert Opinion on Drug Metabolism & Toxicology, 2006. 2(2): p. 231-240.

Ms. nili vasserman

Verified customer

Submitted Jun 21 2019

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