Navigation Links
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 mined using a Bio-Rad Protein Assay Kit. Plant samples were diluted to 400 g/ml of extractable protein in buffer. Two milliliters of diluted extract were used for each reading. A dilution series (0.10.4 g/ml) of purified rGFP-S65T protein (Clontech), was made in either extraction buffer or 400 g/ml untransformed callus extract.

The VersaFluor fluorometer was fitted with a 490 5 nm excitation filter and a 510 5 nm emission filter so that the reflective face of the filters faced towards the incident beam. The VersaFluor fluorometer was used according to the supplied instructions and blanked using 2 ml of extraction buffer. The range (on medium gain) was set using the highest plant sample. All samples and standards were measured by taking the mean of measurements at 20, 40 and 60 seconds.


Results and Discussion
Figure 1 shows the RFUs for the GFP dilution series, quantified using the VersaFluor fluorometer. The presence of other fluorescent compounds in the plant extracts did not increase the RFUs significantly, indicating that the 10 nm bandpass emission filter adequately omitted fluorescence at other wavelengths.

The curve for RFUs against rGFP-S65T concentration was linear over the range of plant GFP concentrations measured. Measurements were made after the optical sensor in the VersaFluor had stabilized. An example of stabilization of the fluorescence readings is shown for a 0.2 g/ml rGFP-S65T protein standard in Figure 2. For most readings, the signal stability varied less than 10% of the original value over 180 seconds. Plant samples were then read as for the GFP-S65T dilution series. GFP concentration was extrapolated from the standard curve corresp onding to the dilution series in untransformed calli extract to account for the background fluorescence. GFP levels in calli expressing Ubi-sgfpS65Tnos3 ranged up to 0.525 g rGFP-S65T per mg of extractable protein.

The ability to quantify GFP in extracts from transgenic organisms facilitates the comparative assessment of expression levels directed and influenced by attached genetic sequences, such as promoters, introns and terminators. The ability to test gene sequences in this way is a prerequisite for the correct deployment of useful gene sequences in transgenic plants.


Reference
1. Heim, R., Cubitt, A. B. and Tsien, R. Y. Nature, 373, 663-664 (1995).


back to top
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Mx4000 Multiplex Quantitative PCR System*,PPP
2. Improved Quantitative Selectivity of Clenbuterol in Human Urine using High Resolution on the TSQ Quantum Mass Spectrometer
3. The LightCycler is the only System for Quantitative PCR that is as Flexible as You are!
4. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
5. Quantitative end-point RT-PCR gene expression measurement using the Agilent 2100 Bioanalyzer and standardized RT-PCR
6. Detection of Genetically Modified Soybean in Processed Foods Using Real-Time Quantitative PCR with SYBR Green I Dye on the DNA Engine Opticon 2 System
7. Qualitative and Quantitative Assessment of Plasmid DNA Isolation, Rev A
8. Quantitative Analysis of Protein-DNA Associations in Vivo Using Real-Time PCR, Rev A
9. Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A
10. Multiplexed Quantitative Peptide Assays for Protein Biomarkers of Cardiovascular Disease in Human Plasma
11. Quantitative Analysis of the Cell Cycle in Imaging Applications
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/24/2016)... , ... June 24, 2016 , ... While the majority ... as the Cary 5000 and the 6000i models are higher end machines that use ... height of the spectrophotometer’s light beam from the bottom of the cuvette holder. ...
(Date:6/23/2016)... Seattle, WA (PRWEB) , ... June 23, 2016 ... ... technology, announces the release of its second eBook, “Clinical Trials Patient Recruitment and ... patient recruitment and retention in this eBook by providing practical tips, tools, and ...
(Date:6/23/2016)... 23, 2016   Boston Biomedical , an ... designed to target cancer stemness pathways, announced that ... Orphan Drug Designation from the U.S. Food and ... cancer, including gastroesophageal junction (GEJ) cancer. Napabucasin is ... inhibit cancer stemness pathways by targeting STAT3, and ...
(Date:6/23/2016)... , June 23, 2016 A person commits a ... crime scene to track the criminal down. An ... Food and Drug Administration (FDA) uses DNA evidence to track ... Sound far-fetched? It,s not. The FDA has increasingly ... support investigations of foodborne illnesses. Put as simply as possible, ...
Breaking Biology Technology:
(Date:4/19/2016)... 20, 2016 The new GEZE ... compact web-based "all-in-one" system solution for all door components. ... or the door interface with integration authorization management system, ... systems. The minimal dimensions of the access control and ... building installations offer considerable freedom of design with regard ...
(Date:4/13/2016)... April 13, 2016  IMPOWER physicians supporting Medicaid patients ... a new clinical standard in telehealth thanks to a ... the higi platform, IMPOWER patients can routinely track key ... body mass index, and, when they opt in, share ... visit to a local retail location at no cost. ...
(Date:3/23/2016)... March 23, 2016 ... Sicherheit Gesichts- und Stimmerkennung mit Passwörtern ... (NASDAQ: MESG ), ein führender Anbieter ... Unternehmen mit SpeechPro zusammenarbeitet, um erstmals dessen ... wird die Möglichkeit angeboten, im Rahmen mobiler ...
Breaking Biology News(10 mins):