Description
Hydroxyproline Assay Kit (BA0219) (BA0219)
The Hydroxyproline Assay Kit (SKU: BA0219) delivers a safe, simple and sensitive means to quantify hydroxyproline and collagen in biologic and cosmetic samples. Hydroxyproline is a modified amino acid found almost exclusively in collagen, the key structural protein of the extracellular matrix, so hydroxyproline content can be used as a proxy for collagen content. In this procedure hydroxyproline is oxidised to a pyrrole ring which then reacts with a dye reagent to yield a pink product measured at 560 nm. The assay uses improved perchlorate-free chemistry, requires only 20 microlitres of sample and has a linear detection range of 0.5 ug/mL to 50 ug/mL hydroxyproline in a 96-well plate. Collagen can be conveniently hydrolysed in Eppendorf tubes and the final incubation performed at 37 degrees C.
| Product Name: | Hydroxyproline Assay Kit (BA0219) |
| SKU: | BA0219 |
| Detection Method: | Colorimetric; read at 560 nm |
| Detection Range: | 0.5 ug/mL to 50 ug/mL hydroxyproline |
| Sample Type: | Biologic and cosmetic samples, serum, cells, urine, tissue lysate, collagen |
| Species Reactivity: | All |
| Assay Time: | Approximately 2 hours |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store kit components at 4 degrees C upon receiving. |
| Shelf Life: | 12 months after receipt |
| Shipping: | Room Temperature |
Hydroxyproline is a unique modified amino acid found exclusively in several animal proteins, the most prevalent of which is collagen. Collagen is a key structural protein of the extracellular matrix, common in connective tissues, and is crucial for medical applications such as tissue transplantation and cell-culture scaffolding as well as in the beauty industry. In this kit, hydroxyproline in the sample is first oxidised to a pyrrole ring, which then reacts with a dye reagent to yield a pink product measured at 560 nm. Because hydroxyproline exists almost exclusively in collagen, its content can be used as a proxy for collagen content.
- Safe: uses an improved perchlorate-free chemistry.
- Sensitive and accurate: uses 20 microlitres of sample with a linear detection range of 0.5 ug/mL to 50 ug/mL hydroxyproline in a 96-well plate assay.
- Convenient: collagen can be hydrolysed in Eppendorf tubes and the final incubation performed at 37 degrees C.
- Direct assays of hydroxyproline and collagen in biologic and cosmetic samples.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | This kit detects free hydroxyproline, so any collagen-containing samples must be hydrolysed prior to assay: combine 50 microlitres of sample with 50 microlitres of 10N NaOH in Eppendorf tubes, heat at 100 degrees C for 1 hour (solid samples may need longer), allow to cool, neutralise with 50 microlitres of 10N HCl, dilute 1:1 with 150 microlitres of deionised water and centrifuge for 2 minutes to collect liquid and pellet debris. |
| 2 | For biological fluid samples such as serum, cells, urine and tissue lysate, first centrifuge to remove particulates; a serial dilution may be needed to bring the sample within the detection limit. |
| 3 | Prepare a 50 ug/mL Hydroxyproline Standard by mixing 20 microlitres of 1 mg/mL Hydroxyproline Standard with 380 microlitres of distilled water, then prepare standards in 1.5 mL centrifuge tubes as described in the standard preparation table. |
| 4 | Transfer 20 microlitres of standards and samples into separate wells of a clear flat-bottom 96-well plate. |
| 5 | For each well mix 8 microlitres of Reagent A with 90 microlitres of Oxidation Buffer, add 90 microlitres of this mix to each well, tap the plate to mix and incubate 10 minutes at room temperature. |
| 6 | Add 90 microlitres of Reagent B to all wells; the wells will become turbid, so pipette up and down until the turbidity dissipates. |
| 7 | Incubate for 90 minutes at 37 degrees C in a plate reader or incubator. |
| 8 | Read optical density at 560 nm, using the 90-minute time point for analysis. |
Subtract the blank value (#4) from the standard values and plot the delta-OD against standard concentrations. Determine the slope and calculate the hydroxyproline concentration as: [Hydroxyproline] = [(ODsample - ODblank) / Slope (ug/mL-1)] x n (ug/mL), where ODsample and ODblank are the OD readings of the Sample and Blank respectively and n is the sample dilution factor. Note: the dilution factor n for the hydrolysis protocol is 6 (50 uL sample + 50 uL 10N NaOH + 50 uL 10N HCl + 150 uL dH2O). Conversions: 50 ug/mL equals 5 mg/dL or 50 ppm. Hydroxyproline constitutes 13% of total collagen weight on average, so 1 ug/mL hydroxyproline is equivalent to 1/0.13 or 7.69 ug/mL collagen.
| Component | Quantity | Storage |
| Reagent A | 1 mL | 4 degrees C |
| Reagent B | 10 mL | 4 degrees C |
| Oxidation Buffer | 10 mL | 4 degrees C |
| Hydroxyproline Standard | 200 uL | 4 degrees C |
