Dessicate sample in a drying oven until all water has been removed(Can measure wet weight, but dry is more accurate). Add 50 µL 6N NaOH for every 150 mg of sample to be hydrolyzed in an Eppendorf tube. Heat in a heat block at 100°C for 1-2 hours. Allow tube to cool. Add equivalent 10N HCl to neutralize NaOH. Add a volume of deionized water equal to the amount of NaOH and HCl added. (A detailed hydrolysis procedure is in the product datasheet).
We have found that the hydroxyproline standards and blank are very similar whether or not they undergo hydrolysis. Therefore, the kit does not require hydrolysis treatment of the hydroxyproline standard.
The incubation can be set to 45°C for 50 minutes. The protocol states to use 37°C because then it can be run in a plate reader or a tissue culture incubator. Some plate readers may not heat to 45°C.
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