![]() The blue emitting variants generally require the use of the 360/40 excitation filter in conjunction with the 460/40 emission filter.Īs noted above, many of the GFP variants utilize filters that are provided standard with the FL600. ![]() Note that although the 508/20 emission filter is closer to the emission peak of the sample, overlap with the 485/20 filter precludes the use of that filter, necessitating the use of the 530/25 nm filter. The red-shifted mutants have been developed for use with the standard "fluorescein" filter set and as such utilize the 485/20 excitation and 530/25 emission filter set. Data was derived from reported spectra in the literature. Representative excitation (dashed lines) and emission (solid lines) spectra of the three basic GFP variants. Excitation and emission spectra of EGFP, wtGFP, and EBFP. Note that although the peak for excitation of wtGFP, GFPuv, and the Stemmer mutant is closer to the 400/30 excitation filter, the 360/40 filter that is a standard filter for the FL600 provides quite adequate fluorescent signal.įigure 1. Table 1 indicates the possible filter sets that would be recommend for the use of the FL600 in measuring the fluorescence of different GFP variants. ![]() # Note that the 400/30 excitation is recommended as being slightly superior. Excitation and emission data of GFP variantsĤ00/30 # Note that the center wavelength and bandpass are indicated. The wild type like variants have their primary excitation peak centered on 395 nm, with an emission peak at 509 nm while the blue emitting mutants generally have an excitation peak at around 380 nm and an emission peak near 460 nm (Figure 1). As demonstrated in Figure 1, the red-shifted variants, typified by EGFP, have a single excitation peak centered at about 488 nm, with an emission peak wavelength of 509 nm. In terms of filter sets for the FL600 microplate fluorescence reader, the most commonly used mutations to the wtGFP protein can be categorized into three groups: (1) the red-shifted variants (2) the wild type like variants and (3) the blue emitting variants. Here we characterize the excitation and emission spectra of some of the commonly used variants of GFP and recommend appropriate filter sets for detection and quantitation using the FL600 microplate fluorescence reader. In order for maximal sensitivity to be obtained when quantitating these GFPs it is important that the most appropriate filter set be used. Several of these variants have different excitation and emission spectra than wtGFP. As a result of the variety of applications several variants form the original wild type green fluorescent protein (wtGFP) have been developed. Green fluorescent proteins are being used for more and more applications in molecular and cellular biology.
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