Hello everyone,

I'm having persistent issues with a colorimetric hydroxyproline assay on decellularized tissue samples (ECM). I'm following protocols based on chloramine-T(oxidation buffer ph is 6 - 6.5) and DMAB (Ehrlich’s reagent), as outlined in the papers by Derek et al., 2017 ("A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich’s Solution Accurately Measures Tissue Collagen Content") and Brown et al., 2001 ("Microplate Assay for the Measurement of Hydroxyproline in Acid-Hydrolyzed Tissue Samples").

Protocol Summary:

• Sample preparation: Dry tissue (10–40 mg) hydrolyzed in 6N HCl at 60 °C for 16–18 hrs.
• Filtration: Filter using a 0.2 μm syringe filter.
• Drying: Hydrolysate dried in a 96-well plate at 60 °C.
• Reconstitution & Oxidation: Reconstituted with water, oxidized with 0.05 M chloramine-T (with the buffer adjusted to pH 6).
• Color development: DMAB reagent added, incubated at 60–70 °C for 60–90 min.
• Reading: Read at 540–560 nm using a microplate reader.

Issue: After adding the DMAB and incubating, I am consistently observing precipitate or cloudiness in the wells, but there is no visible color change, even though all reagents were freshly prepared.

I am also not getting any color change with my standards.

For my standard curve, I am using both marine collagen and a pure hydroxyproline stock (1 mg/mL in 10 mM HCl), with working concentrations of 0, 2, 4, 6, 8, 10 μg/mL, and also at higher concentrations (5, 10, 15, 20, 25 μg/mL). I did not perform the acid hydrolysis step on the pure hydroxyproline standards.

I would greatly appreciate any troubleshooting tips. I'm particularly interested in advice related to:

Has anyone faced a similar issue with this specific protocol or with decellularized tissue samples? Any insights or protocol recommendations from your experience would be a huge help!

Thank you so much for your time and expertise.