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Wavelength Scanning Fluorescent Proteins with EnVision® Multilabel Reader
Sponsored,vendor-submitted protocol   Published in November 2009 (p.37) DOI: 10.2144/000113034

EnVision® Multilabel Microplate Reader, one the most favored multilabel readers in biological assay studies, incorporates monochromator technology in two new options. The Absorbance Monochromator option is capable of performing photometric assays while the Fluorescence Intensity Monochromator option with quadruple monochromator design enables both photometric and fluorescence intensity assays.

The EnVision® with Monochromators measures chromophores or fluorophores at any set wavelength within the operational wavelength range. The range in fluorescence measurements is 230–850 nm and up to 1000 nm in absorbance assays in 0.1 nm increments. The different spectral properties of a sample can be explored by scanning the absorbance, excitation, or emission spectra. Graphical display includes a whole plate view and detailed overlay view option for comparing samples. In result files, the spectral information is attached as graphs or numerical data. Spectral data reduction includes for example determination of the peak wavelength and reporting the response at the specified or peak wavelength.

Materials

2103 EnVision Multilabel Plate Reader with Monochromator Option
(2104 EnVision Multilabel Plate Reader with 2104-8520 Monochromator option)

Recombinant Wild-type GFP
(rGFP, #8360-2; Clontech, Madison, WI, USA)

Enhanced GFP
(rEGFP, #8365-1; Clontech)

Black 96-well OptiPlate
(#6005279; PerkinElmer)

Example Assay

Spectral scans were performed from two fluorescent protein variants. Recombinant wild-type GFP and enhanced GFP were both diluted to 10 µg/ml. GFP samples (200 µl) were pipetted into a black 96-well OptiPlate for measurement of spectral properties. Measurement time was set to 500 flashes with 1-nm step increments. Emission wavelength was set to 565 nm while scanning the excitation spectra and excitation wavelength was set to 425 nm while scanning the emission spectra. Obtained clear and smooth spectra are shown in Figure 3. The excitation peak of EGFP is red-shifted to 485 nm, while the emission maintains the same wavelength properties in both variants.







Correspondence

Address correspondence to PerkinElmer, Inc., 940 Winter Street, Waltham, Massachusetts 02451, USA, http://www.perkinelmer.com/envision2, phone: (800-762-4000 (US & Canada) or (+1) 203-925-4602), email: [email protected]

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