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Determination of Soluble and Total Collagen

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by

Natacha Roussel

on 10 February 2016

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Transcript of Determination of Soluble and Total Collagen

What is the Collagen ?
Determination of the Collagen
Main structural protein in the
extracellular space.
Mechanical resistance to stretching.
Substance that holds the whole
body together.
Several type of collagen.

Usefullness
medicine
agri-food industry
cosmetology
Pulverized meat samples (4 g).

Suspend with 12 ml of strength Ringer’s solution.


Second step
Heat in the water bath
77 °C
70min
Homogenize
10 revolutions
Every 10 min
Let for 30 min at room temperature
Third step
Centrifugation
2°C
6000 X G
10 min
Remove supernatant into 50-ml screw top test tubes with soluble collagen
8 mL
Suspend with strength Ringer’s solution
Fourth step
Remake the centrifugation and the supernatant removal
Add dd H2O into test tube
8 mL
Pour into 50-ml screw top test tubes
Add concentrated hydrochloric acid to centrifuge tubes
10 ml
Pour into test tubes
Add concentrated HCl to supernatant tube
16 ml
fifth step
Separation of soluble and Insoluble Collagen to obtain hydroxyproline
First step
Interest
Meat tenderness
depends on many factors
- race
- age
- sex
- rearing conditions

Medical diagnosis
bone disease


First step
Add aliquots of sample into screw top tubes
1 mL

Add isopropanol to tube and vortex
2 mL
Second step
Add oxidant solution in the tube
1 mL

Vortex

Let at room temperature
for 4 min

Third step
Add Erlich’s reagent to the tube and close this one.
2 mL

Vortex.
Fourth step
Heat tubes in the water bath
25 min
60° C

Cool tubes in running tap water
4-5 min
Fifth step
Vortex

Measure absorbance
558 nm
Determination of hydroxyproline
Collagen Analysis Results
Exemple of regression
Determination of hydroxyproline content
Convert hydroxyproline to collagen (μg collagen/g of sample)
multiply the supernatants by 7.52

multiply the residuals by 7.25
masse of hydroxyproline x dilution factor
masse of sample

NH
HO
HO
O
Alternative methods
Chemical methods
Physical methods
Histochemical methods
Immunochemical methods
Sixth step
Add methyl red indicator
7-10 drops
mix
neutralize the solution
5.0 N NAOH to a yellow endpoint.
filter through Whatman N°1
dilute
supernatants to 100 ml
residuals to 500 ml

With dd H2O
autoclave
12 h ,120-127°,20 + 2 lbs
Add carbon decolorizing agent
200 mg to the supernatant
900 mg to the residuals
mix and filter through Whatman N°1
Full transcript