Abstract
This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction): NH4+ + hypochlorite + OH- + phenol → indophenol The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although it is also valid for other plants species.
Keywords: Ammonium, Indophenol blue, Spectrophotometry, Plant
Materials and Reagents
Equipment
Procedure
Data analysis
Subtract the blank absorbance from the absorbance of every sample and calculate the ammonium concentration with the standards calibration curve. Finally, express the ammonium content in a fresh weight basis. These calculations might be also done directly by the microplate reader software assigning to every well a category for example, blank, standard or sample.
Notes
Recipes
Acknowledgments
This protocol is adapted from Sarasketa et al. (2014) and based on the methodology reported by Solorzano (1969). This work was supported by the Basque Government (IT526-10), the UPV/EHU (EHUA14/14), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007–2013) under REA grant agreement number 334019 and MINECO (BIO2014-56271-R).
References
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