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hydroxyproline assay to determine collagen content

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hydroxyproline assay to determine collagen content

Postby Stip » Wed Aug 03, 2011 5:55 am

Hi,

I am trying to analyse the hydroxyproline to determine collagen content in rat heart samples, I am following Reddy and Enwemeka 1996 paper but unable to get a proper standard curve, even my blank (water) looks similar to my highest standard concentration (2­­0ug). Not sure where I am going wrong, wonder if anyone tried this assay and if so can help me with a protocol please? thanks in advance.

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Postby JackBean » Wed Aug 03, 2011 6:03 am

try to describe the protocol, maybe will we be then able to give some advice.
Didn't you try the authors?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: hydroxyproline assay to determine collagen content

Postby Stip » Wed Aug 03, 2011 6:15 am

thanks Jack, here is my protocol:

50ul of test samples containing Hyp (10ug/ml) and aliquots of std hyp (2-20ug) prepared from 10mg/ml stock, this is then hydrolysed with 50ul of 2N NaOH (2N final conc) at 120 degree C for 20 min. To this add 450ul of Chloramine T and oxidise at RT for 25 min. 500ul Ehrlich's reagent added and chromophore developed by incubating at 65 deg for 20 min. Absorbance was read at 550nm.

I did not try the authors yet.

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Re: hydroxyproline assay to determine collagen content

Postby ucsfmed2012 » Thu Oct 27, 2011 9:30 am

Hello Stip,

I have an adjusted protocol for intervertebral discs. This assay estimates the amount of hydroxyproline, which has been shown to account for approximately 14% the amino acids in collagen.

Materials:
Hydroxyproline Standard - 1-5 µg/2 0.001N HCl ml.
Buffer-0.5 g citric acid monohydrate, 0.12 ml glacial acetic acid, 0.82 g sodium acetate anhydrous, and 0.34 g sodium hydroxide are combined and the final volume is brought to 10 ml with distilled water. Make sure the pH is 6.0. Store in the refrigerator with aluminum foil as a cover.
Chloramine T solution-2mL (0.05M in DIH2O) + 3mL Cellosolve + 5ml buffer
Perchloric Acid (3.15M)
p-Dimethylaminobenzaldehyde in cellosolve (2g/10ml)

100ul of supernatant from each sample, a series of standards, and a blank (0.001 N HCl) were placed in 96well clearbottom microplate.

Hydroxyproline oxidation was initiated by adding 50ul chloramine T solution to each well. Agitate and let stand for 20 minutes at room temperature.

50ul perchloric acid is added to each tube to destroy chloramine T in the same sequence as before. Shake and let stand for 5 minutes.

Add 50ul p-dimethylaminobenzaldehyde solution and mix until no schlieren can be seen (Dissolution of the p-dimethylaminobenzaldehyde may be expedited by heating on a hot plate) then placed in a 60C water bath for 20 min (at this point observe the colorimetric change of the solutions to a red spectra). Cool at rt for 5 min. The fluorescence readings are taken in the MR600 micro-plate reader at 585 nm.

Please let me know if anything is unclear.
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Re: hydroxyproline assay to determine collagen content

Postby enndiaye » Fri Nov 04, 2011 4:24 pm

Hi,


This is a kit that works well :Catalog number K555-100 from Biovision. It's a colorimetric assay similar to yours. The standards work nicely, although the range could be extended if needed.
Good luck !
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Postby Jfangy8 » Tue Aug 20, 2013 12:53 am

Hello ucsfmed2012,
How do you treat your samples(100ul of supernatant from each sample)? Do you add 100 μl
concentrated HCl (~12N, not provided) in a pressure-tight, teflon capped vial and hydrolyze at
120°C for 3 hours?

Thanks!
L.
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