[Histonet] RE: measuring collagen

[Boyce, Amanda (NIH/NIAMS)]

Our lab uses a hydroxyproline assay.  I've never used it, so I can't troubleshoot it for you, but here's the protocol:

Acid hydrolysis
	*	 Papain digest and 12N HCL 1:1
	*	110 overnight in sealed glass ampules
	*	dry sample 24-48 h in vacuum-pump with NaOH
	*	resuspend in 1 ml assay buffer, vortex, overnight at 4, vortex again
	*	store at -20 or assay

Hydroxyproline assay
Reagents:
	*	stock buffer: 50 g citric acid monohydrate, 12 ml glacial acetic acid , 120 g sodium acetate trihydrate, 34 g sodium hydroxide ad 1 liter, pH 6.0, store at 4
	*	assay buffer: 1:10 dilution of stock buffer
	*	chloramin-T-reagent: 0.3525g Chloramine T dissolved in 5.175 ml water, add 6.5 ml m-propanol and 13.325 ml stock buffer, make fresh
	*	Dimethylaminobenzaldehyde reagent: 3.75 g dimethylaminobenzaldehyde in 15 ml n-propanol, add 6.5 ml perchloric acid, make fresh
	*	Standard: 1 mg/ml hydroyxproline, store -20
Method:
	1.	standard dilutions: 0, 0 , 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8 ,10 µg/ml
	2.	sample dilutions: 1:5, 1:10, 1:20, 1:40, 1:80,1: 100
	3.	200 µl sample/standard in eppendorf
	4.	add 100 µl Chloramine T, pipett mix 20 min room temp
	5.	add 100 µl dimethylaminobenzaldehyde, pipet mix, 15 min 60 water bath
	6.	300 µl in 96 well plate and read immediately 540 nm

--------------------------
Amanda Taylor Boyce, Ph.D.
Postdoctoral IRTA Fellow
Cartilage Biology and Orthopaedics Branch
National Institute of Arthritis and Musculoskeletal and Skin Diseases
National Institutes of Health
Bldg. 13, Rm. 3W17
Bethesda, MD 20892-5755
Phone: 301-451-6860
Fax: 301-480-4315
Email: boycea <@t> mail.nih.gov

> Message: 6
> Date: Mon, 7 Nov 2005 07:00:09 -0800 (PST)
> From: Niloufar Fozouni <niloof <@t> yahoo.com>
> Subject: [Histonet] measuring collagen
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <20051107150019.64420.qmail <@t> web35602.mail.mud.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
> 
> Hello,
> 
> Does anybody know, how I can measure collagen content
> in cartilage?
> Thank you.
> Niloo
> 
>