Collagen is the one of the main components of extracellular matrix, and accounts for about 30% of the whole human protein. Recent studies, the production of collagen are reduced in aged skin, the degradation and the accumulation of collagen found in a particular disease, such as has been shown. Therefore, it has become important in accurately quantifying technology health maintenance and disease diagnosis collagen.
Fig. 1. Principle of collagen determination
Collagen contains approximately 30 % Glycine (Gly) of whole amino acid residues in each molecule, and constitutes a repeated amino acid sequence, -Gly-X-Y- in which Pro or Hyp frequently appears at the X or Y position. The proposed assay method takes an advantage of this unique sequence of collagen as well as the remarkable specifi city of bacterial collagenase that cleaves all collagen types at the position of N-terminal side of the Gly residue. Therefore, abundant N-terminal Gly-containing peptides such as Gly-Pro-Hyp, Gly-Pro-Ala, Gly-Pro-Pro, and Gly-Pro-Z (Z is other amino acid or oligopeptide) are produced from one molecule of each collagen, and greatly amplifi ed fl uorescence signals from these products are obtained by the reaction with 3,4-Dihydroxyphenylacetic acid (3,4-DHPAA). This kit is a highly specific and sensitive method for the assay of whole collagen in biological samples using a fluorogenic reagent, 3,4-Dihydroxyphenylacetic acid (3,4-DHPAA). The 3, 4-DHPAA reagent can selectively detect N-terminal Gly-containing peptides.
This kit can quickly measure the collagen in the food and cosmetics. Compared to the existing hydroxyproline method, there is no need of hydrochloric acid hydrolysis, it can be determined in a safe and simple procedure with a small amount of sample. This kit is suitable for quality control and product development during the manufacturing process.
♦ Simple procedure: takes only 2 - 3 hours
♦ Fast and convenient
♦ The kit can measure 96 tests.
|Enzyme Reagent (Collagenase)||200 μL||1|
|Standard solution(500 μg/mL Collagen)||300 μL||1|
|Buffer A||30 mL||1|
|Fluorescence Reagent (3,4-DHPAA)||500 μL||1|
|Buff er B||15 μL||1|
|NaIO4 solution||5 mL||1|
Required but not provided
• PBS(-) (without Mg and Ca)
• 1.5 mL and 500 μL micro-test tube
• 96 well Black Microplate
• 96 well Fluorescent Microplate Reader (Ex: 375 nm/Em: 465 nm)
※ Please prepare a microcell cuvette if you are using a fluorescence spectrophotometer.
Measurement of cell layer collagen in a rabbit chondrocyte
Collagen Quantitation Kit
 Selective and sensitive determination of peptides using 3, 4-dihydroxyphenylacetic acid as a fluorogenic reagent. Hasina Yasmin, Takayuki Shibata, Mohammed Shafikur Rahman, Tsutomu Kabashima, Masaaki Kai, Analytica Chimica Acta, 721 (2012) 162-166
 Amplified and selective assay of collagens by enzymatic and fluorescent reactions. Hasina Yasmin, Tsutomu Kabashima, Mohammed Shafi kur Rahman, Takayuki Shibata, Masaaki Kai, Scientifi c Reports (May 2014) | 4 : 4950 | DOI: 10.1038/srep04950
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.