Collagen Type Ⅰ,Human,ELISA Kit,with Pepsin (96well)

Product#: EC1-E205
$1,096.00
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Collagen Type Ⅰ,Human,ELISA Kit,with Pepsin (96well) 


Cat. No. EC1-E205


Store at 4°C
Size 1kit



Description

This kit is designed to quantify collagen in various sources such as cell media, ECM (Extra Cellular Matrix) of culture cell and tissue because the kit
Sensitivity : 0.02ug/ml
Range: 0.02-40ug/ml

Type 1 collagen is the most abundant protein in the connective tissues, especially in tendon, skin and bone. This kit is designed to quantify collagen in various sources such as cell media, ECM (Extra Cellular Matrix) of culture cell and tissue because the kit detects atelo-collagen which is prepared by pepsin digestion.

Features

  • Short assay time (2 hours 15 minutes).
  • Collagen pre-coated microtiter-plate.
  • Simultaneous assay of many samples (Assay maximum is 40 samples per 1 kit in duplicate).
  • No need of special machines and equipments because of non-isotope assay.
  • Partitional use because of split type (8 wells/strip).
Principle(Competitive EIA)
This assay is a competitive EIA in which polyclonal antibody to human type 1 atelo-collagen is used.

Mixture of sample contained atelo-collagen and biotinylated anti-atelo-collagen antibody is added to a well of microtiter-plate on which purified atelo-collagen is immobilized.

After washing, add peroxidase labeled avidin. The avidin reacts with the biotinylated antibody on the microtiter-plate. After washing, add TMB (3, 3’, 5, 5’-tetramethylbenzidine) which is a substrate of peroxidase.

After reaction, measure the absorbance of the color density of the reaction mixture at 450 nm.

In proportion to increase the collagen concentration of samples, the color density decreases since the amount of biotinyleted antibody and peroxidase labeled avidin decreases.
EC1-E205_fig1.jpg

Preparation of equipments and reagents
  • Micropipettes and tips (10~100 μL、100~500 μL) 
  • 1.5mL tubes
  • Measuring cylinder (500 mL)
  • Distilled or deionized water
  • Microtiter-plate shaker
  • Microtiter-plate reader capable of reading absorbance at 450 nm
  • 50mM Acetic acid solution (for preparation of assay sample of extra-cellular matrix)
  • Neutralizing solution (200 mM Tris, 150 mM NaCl)
Preparation of assay samples
  • Cell culture:Quantification of collagen in culture media
When collagen production cell (fibroblast) is cultured, both pro-collagen and collagen (mature type) are secreted into culture media. Collagen in the culture media can be quantified by direct (It is no need to digest by pepsin). If the concentration of collagen is high, dilute the media by Diluent A, PBS or TBS.
  • Cell culture:Quantification of collagen in extra-cellular matrix (ECM)
When collagen production cell (fibroblast) is cultured in petri-dish, collagen in culture
media deposit as ECM. The deposited collagen can be quantified as follows.
  1. Prepare pepsin solution (Dissolve pepsin powder into 50 mM acetic acid solution to final concentration 0.1 mg/mL).
  2. Remove the culture media from the petri-dish and detach cells from the bottom of the petri-dish using a cell-scraper.
  3. Add equal volume of the pepsin solution to the petri-dish (i.e. : When cells proliferate to 100% confluent, 0.5 mL for a 24 wells-plate, 5 mL for a 6 cm petri-dish).
  4. Shake for overnight at 4˚C.
  5. Centrifuge for 10 miniutes at 10,000 g.
  6. Recover the supernatant.
  7. Add 1/3 volume of neutralization solution (200 mM Tris, 150 mM NaCl) to pepsin solution and mix well .
  8. The mixture can be used as assay sample. 

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