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Evaluating GFP Assays
With GFP (Green Fluorescent Protein) assays, the fluorescent substance is not a dye, but
a protein. Cells can be transfected with a target gene together with the GFP-producing
gene. Transfected cells produce the fluorescent protein, which can be detected. The
fluorescence shows you the success of the transfection experiment. For detailed
information on GFP assays, refer to the application note Monitoring transfection
efficiency by green fluorescent protein (GFP) detection with the Agilent 2100 Bioanalyzer.
For a detailed description on how to evaluate the results using histograms and regions,
refer to “Using Histograms for Evaluation” on page 212 and “Using Dot Plots for
Evaluation” on page 233.
Gating direction
The GFP has a green fluorescence (absorption in the blue). Because the reference dye
(CBNF) fluoresces in the red, the gating direction is from red to blue. CBNF stains living
cells, so you can detect living, GFP-positive cells.
Histogram evaluation
The two histograms displaying the results of the assay are related to CBNF (red
fluorescence) and GFP (blue fluorescence). High fluorescence values in the red
histogram indicate a staining with CBNF, which is associated with living cells. See the
following image as example.
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