Product nameHydroxyproline Assay Kit (Colorimetric)
Sample typeUrine, Serum, Other biological fluids, Tissue Homogenate
Range0.1 µg - 1 µg
Assay time3h 00m
Species reactivityReacts with: Other species, Mammals
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.
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
Storage instructionsStore 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
RelevanceHydroxyproline, 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.
ab222941 has been referenced in 2 publications.
- Yang HY et al. Dendropanax morbifera Ameliorates Thioacetamide-Induced Hepatic Fibrosis via TGF-ß1/Smads Pathways. Int J Biol Sci 15:800-811 (2019). 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). PubMed: 30588110