Type
Colorimetric assay
Description
Now collagen can be easily determined by any biochemical laboratory!
The QuickZyme Total Collagen assay kit is based on the quantitative colorimetric determination of hydroxyproline residues, obtained by acid hydrolysis of collagen. Usually for this analysis upon hydrolysis removal of HCl is needed, which is a time-consuming step and/or needs special equipment. The QuickZyme Total Collagen assay is the only hydroxyproline-based assay which does not need this laborious step, resulting in a fast (<2 hrs) and easy (96-well plate format) assay following the hydrolysis step.
Collagen is one of the main components of extracellular matrix. Dysregulation in collagen production results in pathologies such as fibrosis (too much collagen), or osteoarthritis (too little collagen). Therefore measurement of collagen production is important in many disease related studies.
The QuickZyme Total Collagen assay is based on the detection of hydroxyproline. Hydroxyproline is a non-proteinogenic amino acid, which in mammals occurs in elastin and collagen. Its presence is mainly limited to the triple helix of collagen, where its presence increases the triple helix stability. Hydroxyproline is formed posttranslationally from specific proline residues by action of the enzyme prolylhydroxylase. Hydroxyproline in tissue hydrolysates can be used as a direct measure of the amount of collagen present.
The measurement of collagen is started by complete hydrolysis of tissue samples in 6M HCl at 95°C. In the hydrolysate hydroxyproline residues are quantified using a modification of the method described by Prockop and Udenfriend (Anal. Biochem., 1960, 1: 228-239). The assay measures the total amount of hydroxyproline present in the sample, which represents all the types of collagen present in the sample without discriminating between the types of collagen and between procollagen, mature collagen and collagen degradation products.
The assay is simple and results in a chromogen with an absorbance maximum at 570 nm. The assay is developed in such a way that it doesn’t need the drying step following acid hydrolysis for which often special equipment is needed.
Applications
Tissue, Cellular extracts, Tissue homogenates
Sample Requirements
50 - 500 µl
Shipping
At ambient temperature. Upon receipt, store the product at the temperature recommended below.
Storage/Expiration
Unopened kit: Store at room temperature (RT) in the dark. Do not use kit components past kit expiration date.
Opened kit: reconstituted reagents: The opened collagen standard and assay buffer should be stored light protected at 4°C. The other opened reagents should be stored light protected at RT and are stable for at least 1 month. The reconstituted detection reagent (A+B) should be used on the day of reconstitution.
Calibration Range
0 - 300 µg/ml
Limit of Detection
2.5 µg/ml