hydroxyproline assay to determine collagen content

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    • #15242
      Stip
      Participant

      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.

      stip

    • #105817
      JackBean
      Participant

      try to describe the protocol, maybe will we be then able to give some advice.
      Didn’t you try the authors?

    • #105818
      Stip
      Participant

      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.

      stip

    • #107345
      ucsfmed2012
      Participant

      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.

    • #107617
      enndiaye
      Participant

      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 !
      E.

    • #114206
      Jfangy8
      Participant

      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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