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] = [(OD_sample - OD_blank) / Slope (ug/mL-1)] x n (ug/mL), where OD_sample and OD_blank 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

Neuroimmunologie: Das Immunsystem des ZNS

Interleukin-8-Signalisierung

Waardenburg-Syndrom und Klein-Waardenburg-Syndrom

Hydroxyproline Assay Kit (BA0219)

Description