描述
To efficiently deliver and monitor expression of your gene in live cells, choose a Lentivirus expression vector to fuse your protein with Lumio and V5 epitope tag. Lumio technology is designed for simple fluorescent detection of recombinant protein. The Lumio tag is a six amino acid sequence that binds a fluorescent substrate, allowing you to visually detect your protein of interest in mammalian cells. Choose from either the C-terminal or N-terminal fusion tagged vectors for optimal expression. These Lentiviral Lumio Reporter vectors are configured for making high-titer Lentiviral virus preps for efficient viral delivery to both dividing and non-dividing cells and selection with blasticidin.The pLenti6.2/C,N-Lumio/V5-DEST vectors provide:
CMV promoter for high-level expression of your gene
PGK promoter for long-term stable expression of the Blasticidin (bsd)-resistance marker
Lumio tag for accurate in vivo and in vitro protein detection
Sites for rapid recombinational cloning using Gateway Technology
Lumio技术的优势
The Lumio System is based on the FlAsH (Fluorescein Arsenical Hairpin) technology which uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif (i.e. Lumio tag) (Griffin et al., 1998). Using the Lumio Technology and the Lumio In-Cell Labeling Kits for fluorescence labeling of recombinant proteins provides the following advantages:
Small size of the Lumio tag (6 amino acids, 585 Da) is less likely to interfere with the structure or biological activity of the protein of interest
Lumio Labeling Reagents are membrane-permeable and readily cross the cell membrane, allowing labeling and detection of recombinant proteins in live mammalian cells
Lumio Labeling Reagents bind the Lumio tag with high specificity and high affinity (nanomolar or lower dissociation constant), allowing targeted labeling of the protein of interest
Lumio Labeling Reagents become strongly fluorescent only upon binding the Lumio tag, allowing specific detection of Lumio-tagged proteins
Lumio系统的组成
The Lumio System consists of two major components:
The tetracysteine Lumio tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pLenti6.2/Nor C-Lumio/V5-DEST vector. When fused to a gene of interest, the Lumio tag allows the expressed fusion protein to be specifically recognized by a biarsenical labeling reagent.
A biarsenical labeling reagent, Lumio Green, which becomes fluorescent upon binding to recombinant proteins containing the Lumio tag. LumioGreen Labeling Reagent is supplied pre-complexed to the dithiol EDT (1,2-ethanedithiol), which stabilizes and solubilizes the biarsenic reagent.
Tetracysteine Motif The Lumio Reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-XaaCys-Cys where Cys equals cysteine and Xaa equals any amino acid other than cysteine. This motif is rarely seen in naturally occurring proteins allowing specific fluorescence labeling of recombinant proteins fused to the Lumio tag. In the Lumio System, the optimized Cys-Cys-Pro-Gly-Cys-Cys tetracysteine motif is used as this motif has been shown to have a higher affinity for and more rapid binding to biarsenic compounds as well as enhanced stability compared to other characterized motifs (Adams et al., 2002).
在胶中检测
Lumio For sensitive and specific in-gel detection of Lumio-tagged fusion proteins, we recommend the Lumio Green Detection Kit available (see page 25 for ordering). The Lumio Green Detection Kit enables immediate visualization of Lumio-tagged proteins in polyacrylamide gels using a UV transilluminator or a visible light laser-based scanner and without the need for staining or western blotting. In addition, the BenchMark Fluorescent Protein Standard (see page 25 for ordering) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio Green Detection Reagent.
