Key facts
Detection method:Colorimetric,
Sample types:Urine, Tissue Homogenate, Serum, Other biological fluids,
Assay type:Quantitative,
Range:0.1 - 1 µg,
Assay time:3h,
Assay Platform:Microplate reader
Product details:
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 hydroxyproline assay is used to indicate the amount of collagen in a sample, as hydroxyproline is found in collagen, and is only found in small quantities in other proteins.
How the assay works
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.
The hydroxyprolineassay uses an identical assay principle and assay components to our total collagen assay kit
ab222942
, except that the standard curve of this assay is constructed from hydroxyproline, whereas the total collagen assay uses collagen itself, which makes the total collagen assay a more direct way to quantify collagen levels.
Hydroxyproline assay protocol summary
- Add 10 N concentrated NaOH to samples and hydrolyze at 120°C 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 65°C
- Add oxidation reagent mix to dissolve crystalline residue, and incubate at room temp for 20 min
- Add developer and incubate at 37°C for 5 min
- Add DMAB concentrate and incubate for 45 min at 65°C
- Analyze with microplate reader
Getting the best performance from ab222941
We do not offer K555 Hydroxyproline Colorimetric Assay Kit from BioVision. We recommend ab222941 as an alternative.
This product is a replacement for Hydroxyproline Assay Kit K555; this kit does not contain the hazardous Perchloric acid which is present in K555.
The components in this product are exactly the same as in K555, except that the Perchloric acid/Isopropanol Solution component in K555 has been replaced with the Developer component.
If you would prefer to continue to use a kit in the format of K555, then use this product and:
1) In a fume cupboard, mix 1 ml of 70% Perchloric Acid and 5 ml of Isopropanol to produce the Perchloric acid/Isopropanol Solution component previously provided in K555.
2) Then follow the protocol for K555 below, using the components from this product, and the Perchloric acid/Isopropanol Solution that you produced.
Alternative protocol for Perchloric acid/Isopropanol method:
Do not use with the standard format of the kit, use the main protocol for use with the kit
Sample Preparation
: Tissue or protein/peptide samples such as lung tissue should be homogenized in dH2O, using 100 μl H2O for every 10 mg of tissue. To a 100 μl of sample homogenate, add 100 μl concentrated HCl (~12N, not provided) in a pressure-tight, teflon capped vial and hydrolyze at 120°C for 3 hours. In case of urine, hydrolyze urine samples with equal volumes of concentrated HCl (~12 N; i.e. 100 µl Urine + 100 µl HCl) in a pressure-tight, teflon capped vial. Hydrolyze at 120°C for 3 hrs (See note c). Clarify urine samples with activated charcoal by adding 4 mg of activated charcoal. Vortex and centrifuge at 10000 x g for 3 min. to remove precipitate & activated charcoal. Repeat if needed. Transfer 10 μl of each hydrolyzed sample to a 96-well plate and evaporate to dryness under vacuum.
Notes:
For unknown samples, we suggest performing a pilot experiment & testing different sample dilutions to ensure the readings are within the Standard Curve range. Endogenous compounds may interfere with the reaction. To ensure accurate determination of Hydroxyproline in the test samples, we recommend spiking samples with a known amount of Standard (0.4 µg). For sample hydrolysis, polypropylene vials yield best results.
Standard Curve Preparation
: Dilute the Hydroxyproline Standard to 0.1 mg/ml by adding 10 μl of the 1 mg/ml Standard to 90 μl of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 μl into a series of wells to generate 0.2, 0.4, 0.6, 0.8 & 1 µg/well Hydroxyproline Standard.
Reaction
: Add 100 ul of the Chloramine T reagent to each sample and standard and incubate at room temperature for 5 min. Add 100 μl of the DMAB reagent to each well and incubate for 90 min. at 60°C.
Measurement
: Measure absorbance at 560 nm in a microplate reader
Calculation
: Correct background by subtracting the value derived from the 0 Hydroxyproline Standard from all readings (The background reading can be significant and must be subtracted). Plot the Standard curve. Apply the sample readings to the standard curve to get the hydroxyproline amount in the reaction wells (B).
Sample Hydroxyproline concentration (C) = B/V x D ug/µl
Where:
B is the amount of Hydroxyproline from Standard Curve (µg)
V is the sample volume added into the reaction well (µl)
D is the sample dilution factor
Note: For spiked samples, correct for any sample interference by subtracting the sample reading from spiked sample reading.
Related and recommended products
Other biochemical assays to measure the biology of collagen include:
- Soluble Collagen Assay Kit
ab241015
- Collagenase (Collagen Degradation/Zymography) Assay Kit (Fluorometric)
ab234624
- Collagenase Activity Assay Kit
ab196999
- For collagen staining we provide:
- Trichrome Stain Kit (Connective Tissue Stain)
ab150686
- Picro-Sirius Red Stain Kit (Connective Tissue Stain)
ab150681
- Picro-Sirius Red Solution
ab246832
- Picro-Sirius Red Stain Kit (Cardiac Muscle)
ab245887
Other notes
This product is manufactured by BioVision, an Abcam company and was previously called K226 Hydroxyproline Assay Kit (Perchlorate-Free). K226-100 is the same size as the 100 test size of ab222941.
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information-Please refer to protocols
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Hydroxyproline also known as Hyp is a non-standard amino acid with a molecular weight of approximately 131.13 g/mol. It is primarily found in collagen making up about 13% of its structure. Hydroxyproline expression is mainly localized in tissues where collagen is abundant such as skin bone and cartilage. This makes it an important marker in studies related to connective tissue health and integrity and therefore hydroxyproline assays and kits are commonly used for analyzing collagen content in these tissues.
Biological function summary
Hydroxyproline plays a critical role in stabilizing the triple helical structure of collagen. It is not directly encoded by the genetic code but formed by the post-translational modification of proline residues in collagen chains. The presence of hydroxyproline in collagen is essential as it ensures the structural stability and mechanical strength of the collagen thereby supporting tissue integrity. This modification does not occur in isolation and is often part of a complex series of hydroxylation reactions facilitated by enzymes such as prolyl hydroxylase and ascorbate (vitamin C) as a cofactor.
Pathways
Hydroxyproline is integral to collagen biosynthesis and modification pathways. One significant pathway is collagen fibril biosynthesis where hydroxyproline ensures proper folding and assembly of collagen fibers. In the same pathway proteins like lysyl oxidase also interact with hydroxyproline-modified collagen to facilitate cross-linking endowing the collagen fibers with added tensile strength. Another important pathway is the post-translational modification cascade involved in protein maturation where hydroxyproline plays a role alongside other hydroxylated amino acids.
Hydroxyproline levels are implicated in conditions like osteogenesis imperfecta and scurvy. Osteogenesis imperfecta caused by a defect in collagen type I features disrupted hydroxyproline incorporation leading to brittle bones. Hydroxyproline is also connected to scurvy where vitamin C deficiency impairs proline hydroxylation resulting in weakened collagen structures. In these cases monitoring hydroxyproline can assist in assessing disease progression and therapeutic outcomes highlighting its relevance alongside proteins such as collagen types I and III.
