Simple Plex Green Fluorescent Protein Cartridge

Simple Plex Green Fluorescent Protein Cartridge Summary

*The Sample Volume represented is based on the amount of sample incorporated into the reaction after taking into account the assay’s minimum required dilution for a given matrix. Serial dilution may be necessary to achieve some of the final sample volumes represented.

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Preparation and Storage

Background: GFP

The green fluorescent protein (GFP), originally discovered in the jellyfish, Aequorea victoria, is composed of 238 amino acids and has a molecular weight of 27 kDa. The molecular structure of GFP represents a cylinder, comprising 11 strands of beta-sheet outside, a single alpha helical strand containing the chromophore running through the center, and short helical segments on the ends. GFP transduces the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Because of this property, GFP has been widely used as a tracer in various disciplines of biology research, such as fluorescence microscopy, reporter gene expression, and in vivo imaging.

Laboratory mutation of GFP generated the widely used analog GFPuv, which has brighter fluorescence and more desirable expression properties. GFPuv is the Aequorea sequence with three aa substitutions; Phe to Ser at # 99, Met to Thr at # 153, and Val to Ala at # 163. This form of GFP expresses faster and is 18-fold brighter than native GFP; excitation peaks at 395 nm and emission at 508 nm. In the presence of UV light, GFPuv emits a green fluorescent color that can be used to pinpoint locations of various intracellular proteins.

Antibodies directed against GFPuv provide additional tools for confirming GFPuv expression in cells and tissues. During immunohistochemical processing, the use of alcohols and xylene results in bleaching of GFP proteins. Biochemically, detection of GFPuv by Western blot can support the expression of the tagged protein in the target tissue of interest. GFPuv antibodies can also be employed for immuno-precipitation (IP) and ELISA experiments.