you can extract protein with ripa buffer and then run fastin assay for elastin, Sircol assay for collagen(biocolor company has both). for hydroxyproline , sigma has a kit.

if you want i can give protocols for those.

Dear Nasim, Please give me that protocol for spectroscopic method. I want to manually.

1.   The Sircoll Collagen Assay

The Sircoll Collagen Assay is a dye-binding method for the analysis of acid and pepsin-soluble collagens. The Sircol Assay is suitable for monitoring collagen produced in situ or during in-vitro cell culture and in-vitro extracellular matrix, (ECM), formation.

2.   MATERIALS

0.5 M Acetic Acid (100ml)

Add 3,4 ml of 17 M Acetic Acid (100% glacial) in 96,6 ml Water.

0.5 M NaOH (Alkali Reagent) (100 ml)

Dissolve 2 gr NaOH in 100 ml Water.

Saturated picric acid (1.3% in water)

Add 13 gr of picric acid in 1 L deionized water, stir up with and warm on a hot plate, until most of the picric acid is solved. Never let it alone, stir repeatingly. Let cool over night, picric acid will fall out (and form beautiful cristals). Fill the supernatant in another bottle.

Sircoll dye

0.1% Direct Red 80 in saturated picric acid.

3.                           METHOD

1. Upon euthanasia, the mouse is weighed and the weight recorded.

2.  The lungs, liver are perfused with 2-3 mls 1xPBS via the heart.

3. The tissues are homogenized in 1 ml of 1xPBS.

4.  For clearer results the homogenate is spun for 10 minutes at 2000 rpm and the supernate is saved.   The supernate is poured into a 1.5 eppendorf and saved for elisas if needed.

5.   Rat Tail Collagen at concentration 1 mgr/ml is diluted with acetic acid 0.5M to  concentration of:   500            250      125      62,5     31,25 0 μg/ml at final volume of 100 μl.

6.  100µl of each tissue sample homogenate is pipetted into the labeled eppendorf tubes.

7.   Add 1 ml Sircoll dye to each sample and standards. Then mix on a mechanical shaker, rocker or roller equipment for 30 minutes at room temperature.

8.  Spin the tubes at 5000g for 5 minutes.

9.  Drain the unbound dye solution by turning the tube upside down on a tissue, remove any remaining droplets from the top half of the tube with a tissue.  Do not attempt to remove any fluid close to the pellet.

10.   To each tube add 1ml of Alkali reagent.  Vortex well to release the bound dye into solution for 10 minutes. This occurs usually within three minutes.   

11.  Transfer 100 µl of the unbound dye to a 96 well plate and read with a 540nm wavelength. The plate should be read within three hours.

14.  Using the standard curve, calculate the amount of collagen in your tissue sample.

hydroxyproline assay

thanks.

Hi Vabeiryureilai mathipi, can you let me know how to estimate elastin in rat skin