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Quantitative Analysis of GFP in Plant Extracts Using the VersaFluor Fluorometer System

Adrian R. Elliott, Peer M. Schenk and Christopher P.L. Grof
CSIRO Tropical Agriculture, Brisbane, Queensland


Introduction
The Green Fluorescent Protein (GFP) from Aequorea victoria is a useful reporter gene in higher organisms, including plants and animals. The fluorescence emitted is easy to detect and requires no substrate or cofactors. Furthermore, improved GFP versions have been created with higher fluorescence coefficients, improved expression characteristics for eukaryotes, or altered spectral properties. One form, GFP-S65T incorporates a threonine amino acid substitution in place of serine at position 65, which results in red-shifted fluorescence characteristics.1 This form is one that has been proven to be very beneficial for use in plants.

Quantification of GFP is unlike widely used enzyme-based reporters such as β-glucuronidase (GUS) and firefly luciferase (LUC). We show here that fluorescence detection using the VersaFluor fluorometer system with appropriate narrow band filters can be used to detect and quantify GFP-S65T protein levels in plant extracts against a standard curve made from purified GFP-S65T protein.


Method
The test samples consisted of sugarcane cv Q117 callus expressing Ubi1-sgfpS65T-nos3. Approximately 500 mg of tissue was ground in extraction buffer (10 mM Tris-EDTA, pH 8.0, 0.02% [w/v] sodium azide) at 4 C. This extract was centrifuged in a microcentrifuge at 13,000 rpm for 20 min. and the supernatant removed for analysis. Fluorescence microscopy showed that all visible GFP fluorescence was located in the soluble fraction. Protein concentrations of the plant extracts were deter
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