I am trying to measure Ascorbic acid levels in tissue using Omaye et al protocol (Methods in Enzymology, Volume 62, 1979, Pages 3-11). This method is based on reduction of ferric chloride and subsequent detection by 2, 2'-dipyridyl. However, this assay is a test tube based assay. I want to convert this protocol into a plate based assay, however, I could not find any already published protocol for this. Is it possible to do it?
Kamran：You only need to reduce the volume of the regular reaction and use 96 well plate instead. If there was no publication of such a miniature method, you will be the first to do so! Good luck.
How is product formation detected? Is it by a change in absorbance? If so, what wavelength is measured? Absorbance plate readers are not always capable of measuring at wavelengths shorter than 340 nm, unless they are designed for it. Also, polystyrene plates cannot be used for measurements below 340 nm. You can either use acrylic plates, or a quartz plate. Both are quite expensive.
Because the pathlength through the liquid in a 96-well plate is usually less than 1 cm, the sensitivity of a plate-based absorbance assay is less than the sensitivity of an assay performed in a standard 1-cm pathlength cuvette.
Thanks Adam! The method is based on reduction of ferric iron by ascorbic acid followed by formation of a complex of the ferrous iron product and 2,2’-bipyridinie. The absorbance of the resultant product is measured at 525 nm.
Do you have to heat the samples? Plastic plates have a limited heat tolerance. Also, you have to seal the plates to prevent evaporation during heating.
I hope This article will be helping. good luck
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