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Method of quantitative analysis of collagen by means of capillary electrophoresis and set reagent

A capillary electrophoresis and collagen technology, applied in the field of collagen quantitative analysis, can solve the problems of multi-time, total collagen loss, affecting the results of capillary electrophoresis identification and quantification, etc., and achieves the effect of reducing operation time and quantitative time.

Inactive Publication Date: 2010-10-13
黄玲惠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned collagen cleavage process takes a considerable amount of time, prolonging the overall collagen analysis time. In addition, cyanogen bromide is a toxic substance that needs to be specially handled and discarded, and the pretreatment process of extraction and cleavage of collagen will inevitably make the collagen The loss of the total amount of protein affects the results of identification and quantification after capillary electrophoresis

Method used

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  • Method of quantitative analysis of collagen by means of capillary electrophoresis and set reagent
  • Method of quantitative analysis of collagen by means of capillary electrophoresis and set reagent
  • Method of quantitative analysis of collagen by means of capillary electrophoresis and set reagent

Examples

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Embodiment 1

[0046] Example 1: Capillary Electrophoresis Minimum Detection Sensitivity and Standard Curve Analysis

[0047] In order to define the range of the amount of collagen that can be detected by the capillary electrophoresis method of the present invention, and to establish a collagen standard curve (standard curve), different contents of type I collagen were analyzed by capillary electrophoresis, including 276.8, 207.6, 138.4, 83.6, 76.14, 69.21, 55.37, 54.58, 47.46, 41.53, 35.59, 33.22, 27.76, 23.73, 17.79, 13.76, 11.86, 6.29, 4.88, 3.46, 2.76, 1.73, 1.37, 9, 02.7, 0. 0.16, 0.13, 0.09, 0.06, and 0.05 ng of collagen were injected into an amine capillary (amine capillary, inner diameter 50 μm × outer diameter 360 μm × length 65 cm; Beckman, USA) with an injection pressure of 2 psi and an injection time of 6 seconds. , pH2.5 sodium dihydrogen phosphate buffer solution was analyzed by capillary electrophoresis at 25°C, the electrophoresis voltage was -25kV, and the ultraviolet light ...

Embodiment 2

[0049] Example 2: Capillary Electrophoresis Analysis of Various Types of Collagen

[0050] 10 μL of 1 mg / mL purified collagen solution from different sources, including porcine skin type I collagen (extracted and purified in the laboratory Huang-Lee, LLH & Nimni, ME, 1993, Biomed Eng Appl Basis Comm 5:664-675; Huang, LLH et al., 1998, J Biomed Mater Res 39:630-636), calf skin type I collagen, human placenta type III collagen (human placenta type III collagen), human placenta type IV collagen, human placenta type V collagen (Sigma chemical Co., USA), and pig cartilage II Type II collagen (porcine cartilage type II collagen) (Wako Pure Chemical Industries, Japan) was added to 1.5 μL of 1% benzyl alcohol internal control reagent (internal control) and mixed. The mixture was injected into an amine capillary (amine capillary, inner diameter 50 μm×outer diameter 360 μm×length 65 cm; Beckman, USA) with an injection pressure of 2 psi and an injection time of 6 seconds, buffered with ...

Embodiment 3

[0052] Example 3: Specific analysis of collagen by capillary electrophoresis

[0053] 10 μL of 1 mg / mL collagen mixed solution (including type 1, type 2, type 3, type 4 and type 5 collagen) added with benzyl alcohol and 10 μL each of 1 mg / mL albumin ( albumin) and protein mixture (Beckman Cat.No.477436), the latter contains ribonuclease A (ribonuclease A), cytochrome C (cytochrome C) and lysozyme (lysozyme) solution, with 2psi injection pressure and 6 seconds Injection time Inject into an amine capillary (amine capillary, inner diameter 50 μm × outer diameter 360 μm × length 65 cm; Beckman, USA), and conduct capillary electrophoresis analysis in 50 mM, pH 2.5 sodium dihydrogen phosphate buffer solution at 25 °C at the same time, the electrophoresis voltage It is -25kV, and the ultraviolet light absorption wavelength is 214nm, the result obtained after analysis is as follows Figure 8 shown.

[0054] The peaks of benzyl alcohol, collagen, ribonate hydrolase A, albumin, cytoch...

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Abstract

The invention relates to a method of quantitative analysis of collagen by means of capillary electrophoresis and a set reagent. The method for quantitative analysis of the collagen by means of capillary electrophoresis is used for quantifying and identifying the collagen in a sample, comprising at least the following steps: a) dissolving samples containing the collagen to form a sample solution; b) preparing a capillary with an inner tube wall provided with a positive charge layer; c) introducing the sample solution to the capillary containing a buffer solution for analyzing; and d) driving the sample solution to pass through the channel of the capillary. The invention also relates to a set reagent matched with the method. In the invention, the sample containing the collagen does not need to be subject to the preprocessing process of purifying or decomposing the collagen, and capillary electrophoresis analysis is directly conducted, thus saving the quantitative time of the collagen.

Description

technical field [0001] The invention relates to a method for quantitative analysis of collagen, in particular to a method for quantitative analysis of collagen by capillary electrophoresis and a set of reagents. Background technique [0002] In animals, collagen is widely and abundantly present in the extracellular matrix of connective tissue. Collagen is found to be the main constituent of ligaments, tendons, bones, and cartilage, and the above-mentioned tissues are used to support or provide tension; while in the skin, collagen and keratin are responsible for maintaining the elasticity and strength of the epidermis According to the current literature, collagen is divided into more than 20 types according to its structure and amino acid (or amino acid) composition, called collagen families (collagen families), any type of collagen Genetic defects can lead to serious diseases such as osteogenesis imperfecta. Each specific type of collagen molecule is a triple-helical struc...

Claims

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Application Information

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IPC IPC(8): G01N27/447
Inventor 黄玲惠
Owner 黄玲惠