| HOME >> BIOLOGY >> TECHNOLOGY |
Contributed by Jeff Wilson, and Andrew Segal, M.D., Genitrix, LLC, Cambridge, MA
Introduction
The Green Fluorescent Protein (GFP) derived from Aqueorius victoria has
recently become a popular choice as a reporter gene. GFP can be used,
for example, to study promoter function and to track the expression, localization,
and function of proteins to which it has been recombinantly fused.1,2
When stimulated by light the molecule fluoresces without addition of any
cofactors or substrates. For our studies we have used the Enhanced Green
Fluorescent Protein gene (EGFP), a red-shifted variant of the wild-type
protein, as a reporter gene in Jurkat cells. EGFP has an excitation maximum
at 488 nm, and emission maximum at 507 nm. It is very resistant to photobleaching
and is reported to maintain stable fluorescence from pH 7.0 to 11.5.3
In fluorometric assays, purified EGFP can be measured at concentrations
in the low nanogram range.
In recent experiments we have shown that intracellular EGFP can be assayed using a VersaFluor fluorometer. Using the EX490/10 and EM510/10 filter set, we compared pEGFP-n2 transfected Jurkat cells with wild-type Jurkat cells. To approximate the amount of EGFP produced by transfected Jurkat cells, a standard curve with recombinant Enhanced Green Fluorescent Protein (rEGFP) was also generated. The methods and results from those experiments are reported here.
Methods
Jurkat cells (clone E6-1 from ATCC), a leukemic human T-cell line, were
maintained in RPMI 1640 media with 2 mM L-glutamine and supplemented with
2.5 g/l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, in addition
to 10% fe
'"/>
Source:
Related biology technology :
1. Measuring multiple apoptosis parameters with the Agilent 2100 bioanalyzer
2. Measuring Gene Silencing Effects by RT-PCR Without RNA Isolation
3. Measuring RNase Activity A Real-time Kinetic Analysis
4. Measuring siRNA-mediated knockdown of IL-8 mRNA
5. Enzyme Immunoassay for Studying Intracellular Levels of cAMP
6. Cis-Reporting System Monitors Intracellular Calcium Mobilization
7. Cis-Reporting System Monitors Intracellular Calcium Mobilization
8. Enhanced PCR Cloning System
9. Enhanced Amplification of Long Targets with PfuTurbo DNA Polymerase
10. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
11. Enhanced siRNA Delivery and Long-term Gene Silencing
| Copyright © 2003-2012 Bio-Medicine. All rights reserved. ABOUT | CONTACT US | DISCLAIMER | PRIVACY POLICY | TERMS AND CONDITIONS |  |