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Hydroxyproline assay

XZ Xin Zhou
LB Ludvig J. Backman
PD Patrik Danielson
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Cells were treated the same as described in ELISA assay. Supernatants were collected 2 days after treatments. A hydroxyproline assay was used to determine the free hydroxyproline content in each group. This offered further insights on incorporation of free hydroxyproline in collagen synthesis. This assay was performed on hydrolyzed samples using a hydroxyproline assay kit (Abcam, Cambridge, UK). Briefly, supernatants were mixed with concentrated NaOH (10 N) in a tightened screw-capped polypropylene vial and then heated at 120 °C for 60 min. The alkaline lysate was cooled on ice, neutralized, and centrifuged to obtain supernatants. The hydrolysates were dried, oxidized, and finally reacted with 4-(dimethylamino) benzaldehyde. The resultant colored product was detected at 560 nm and found to be proportional to the hydroxyproline present in the sample.

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