Protocol abstract

Nature Protocols 2, 871 - 874 (2007)
Published online: 12 April 2007 | doi:10.1038/nprot.2007.101

Subject Categories: Isolation, purification and separation | Model organisms | Plant biology | Biochemistry and protein analysis

Measurement of reduced, oxidized and total ascorbate content in plants

Kelly M Gillespie1 & Elizabeth A Ainsworth1,2

Ascorbate is one of the major antioxidant metabolites in plant tissues. This protocol describes a microplate-adapted colorimetric ascorbate assay, in which ferric ion is reduced by ascorbate to the ferrous ion. The ferrous ion reacts with alpha-alpha'-bipyridl to form a complex with characteristic absorbance at 525 nm. With the chemical reduction of any dehydroascorbate (DHA) in a sample, total ascorbate can be assayed using the alpha-alpha'-bipyridl method, and DHA can be estimated by subtracting the reduced portion from the total ascorbate pool. The assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. Reduced ascorbate, DHA and total ascorbate of at least 64 experimental samples can be analyzed easily in 1 d.

  1. Program in Physiological and Molecular Plant Biology and Institute for Genomic Biology, University of Illinois Urbana-Champaign, 147 ERML, 1201 W. Gregory Drive, Urbana, Illinois 61801, USA.
  2. USDA ARS Photosynthesis Research Unit, Urbana, Illinois 61801, USA.

Correspondence to: Elizabeth A Ainsworth1,2 e-mail: