Hydroxyproline Assay Kit (Perchlorate-Free)

This kit does not use PCA to detect and quantify Hydroxyproline
Catalog #: K226 | abID: ab222941

Product Details

abID ab222941
Cat # +Size K226-100
Size 100 Reactions
Kit Summary • Detection method- Absorbance (560 nm)
• Species reactivity- Mammalian
• Applications- The assay is useful over the range of 0.1-1 μg.
Detection Method Absorbance (OD = 560 nm).
Species Reactivity Mammalian
Applications Estimation of hydroxyproline concentration in various biological samples
Features & Benefits • Simple procedure; takes less than ~60 minutes
• Convenient and easy to use
• Faster than Perchlorate-Based Assay
• PCA-free
Kit Components • Oxidation Buffer
• Chloramine T Concentrate
• Developer Solution
• DMAB Concentrate (in DMSO)
• Hydroxyproline Standard (1 mg/ml)
• Microplate Sealing Film
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Hydroxyproline ((2S,4R)-4-hydroxyproline, Hyp) is a nonproteinogenic amino acid formed by the enzyme prolyl-4-hydroxylase, which catalizes the post-translational modification of polypeptide proline residues following protein synthesis. In animals, hydroxyproline is found almost entirely in collagen, which contains approximately 12-14% hydroxyproline by mass (the fibrilous protein elastin also contains small quantities of hydroxyproline). Hydroxyproline in tissue hydrolysates is thus a direct measure of the amount of collagen present. Collagen is the most abundant protein in mammals and many diseases are believed to affect collagen turnover, including tumor invasion and metastasis, rheumatoid arthritis, cardiopulmonary fibrosis and muscular dystrophy. The classical assay for determination of Hyp is based upon oxidation of Hyp to a pyrrole intermediate, followed by reaction with Ehrlich’s reagent dissolved in concentrated perchloric acid (HClO4). Perchloric acid is a hazardous material that is both toxic and highly reactive, requiring special handling and waste-disposal protocols. BioVision’s Hydroxyproline Assay Kit II (Perchlorate-Free) uses a proprietary acidic developer solution to accurately measure Hyp in tissue and protein/peptidase hydrolysates without the use of hazardous perchlorates. It is a quick and convenient protocol, yielding a brightly-colored chromophore with an absorbance peak at 560 nm. The assay can detect a minimum of 0.05 µg hydroxyproline per well in a 96-well format and generates colorimetric results that are virtually identical to the classical perchloric acid-based method.

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Our homogenates have been prepared in PBS (not dH2O), would this kit still be suitable?
For K226, it is recommended to homogenize the tissue thoroughly in dH2O before doing the hydrolysis. This is because the salts in PBS may precipitate in the wells during the final reaction step with DMAB. It shouldn’t technically interfere with the assay, but some precipitates may be seen in the wells.
In the protocol, it is recommended to use vials with screwcaps (BV Cat# M1352 and M1353). However, we want to set the assay on a 96-well plate. Do you know what type of plate should we use that would be equivalent to these tubes?
It is not possible to use 96 well plates for the hydrolysis step as you need to do it in a tightly sealed tube to avoid evaporation during high temperature.
How is K226 different from K555?
K226 follows alkaline hydrolysis and it is percholate free where as K555 follows acid hydrolysis protocol. They are both different in the method used to hydrolyze tissue. Alkaline hydrolysis is less time consuming than acid based hydrolysis.
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