pcDNA6.2/nTC-Tag-DEST 载体含有以下元件: Human cytomegalovirus immediate-early (CMV) promoter/enhancer for high-level expression in a wide range of mammalian cells
TC-Tag for C-terminal (pcDNA6.2/cTC-Tag-DEST) or N-terminal (pcDNA6.2/nTC-Tag-DEST) fusion to the gene of interest for fluorescence detection
Two recombination sites, attR1 and attR2, downstream of the CMV promoter for recombinational cloning of the gene of interest from an entry clone
Chloramphenicol resistance gene located between the two attR sites for counterselection
The ccdB gene located between the two attR sites for negative selection
The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
f1 intergenic region for production of single-strand DNA in F plasmidcontaining E. coli
SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the SV40 large T antigen
Blasticidin resistance gene for selection of stable cell lines
The pUC origin for high copy replication and maintenance of the plasmid in E. coli
The ampicillin resistance gene for selection in E. coli For a map of pcDNA6.2/cTC-Tag-DEST and pcDNA6.2/nTC-Tag-DEST, refer to pages 20 and 22, respectively. Tetracysteine 基序 Both the FlAsH-EDT2 and ReAsH-EDT2 reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-Xaa-Cys-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 TC-Tag. In the TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit, 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. Tetracysteine标记技术的优点：
The TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif (i.e. TC-Tag).2 Using the TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits for fluorescence labeling of recombinant proteins provides the following advantages:
Small size of the TC-Tag (6 amino acids, 585 Da) is less likely to interfere with the structure or biological activity of the protein of interest
FlAsH-EDT2 and ReAsH-EDT2 labeling reagents are membrane-permeable and readily cross the cell membrane, allowing labeling and detection of recombinant proteins in live mammalian cells
FlAsH-EDT2 and ReAsH-EDT2 labeling reagents bind the TC-Tag with high specificity and high affinity (nanomolar or lower dissociation constant), allowing targeted labeling of the protein of interest5
FlAsH-EDT2 and ReAsH-EDT2 labeling reagents become strongly fluorescent (green and red, respectively) only upon binding the TC-Tag, allowing specific detection of TC-tagged proteins
FlAsH-EDT2 and ReAsH-EDT2 labeling reagents can be applied sequentially on the same sample, allowing temporal detection of protein turnover and trafficking.
ReAsH-EDT2 labeling reagent can be used for both fluorescence-based microscopy and electron microscopy.
FlAsH-EDT2 labeling reagent provides a superior alternative to yellowfluorescent protein (YFP) when coupled with cyan-fluorescent protein (CFP) for FRET-based cellular analysis. Gateway技术 The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda1 to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. To express your gene of interest in mammalian cells using Gateway Technology, simply:
1. Clone your gene of interest into a Gateway entry vector to create an entry clone.
2. Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway destination vector (e.g.pcDNA6.2/ cTC-Tag-DEST or pcDNA6.2/nTC-Tag-DEST).
3. Transfect your expression clone into the cell line of choice for transient or stable expression of your gene of interest.
For more information on Gateway, refer to the Gateway Technology with Clonase II manual. 检测试剂盒及使用方法 The TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit consists of two major components:
The tetracysteine TC-Tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pcDNA6.2/TCTag-DEST vector. When fused to a gene of interest, the TC-Tag allows the expressed fusion protein to be specifically recognized by a biarsenical labeling reagent. For more information on the tetracysteine motif, see below.
A biarsenical labeling reagent, FlAsH-EDT2 or ReAsH-EDT2, which becomes fluorescent upon binding to recombinant proteins containing the TC-Tag. The FlAsH-EDT2 or ReAsH-EDT2 labeling reagents are supplied pre-complexed to the dithiol EDT (1,2-ethanedithiol) which stabilizes and solubilizes the biarsenic reagents. 检测TC-Tag融合蛋白 Introduction Once you have transfected your expression clone into mammalian cells, you may:
Detect protein expression and localization in live cells by fluorescence microscopy using the TC-FlAsH or TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits. For detailed guidelines and protocols, refer to the TCFlAsH or TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits instruction manual.
Detect protein expression directly in polyacrylamide gels using the Lumio Green Detection Kit. 胶内检测 For sensitive and specific in-gel detection of TC-Tagged fusion proteins, we recommend the Lumio Green Detection Kit available from Invitrogen (LC6090). The Lumio Green Detection Kit enables immediate visualization of TC-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 (LC5928) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio Green Detection Reagent.
Western Blotting You may detect expression of your recombinant fusion protein using the Anti-V5 Antibody (R960-25), Anti-V5-HRP Antibody (R961-25), or Anti-V5-AP Antibody (R962-25) available from Invitrogen. You may use any method of choice to prepare your mammalian cell lysates for Western blot analysis.
We recommend the following guidelines:
If you plan to analyze your samples using the Lumio Green Detection Kit in addition to Western blotting, you will need to prepare your samples using lysis buffer. Lysates containing standard Laemmli SDS-PAGE sample buffer will not be suitable for in-gel detection with the Lumio Green Detection Kit. Refer to the Lumio Green Detection Kit manual for a protocol to prepare cell lysates that are compatible with both in-gel detection and Western blot analysis.
For cells transfected with the pcDNA6.2/nTC-Tag-p64 positive control vector, you will need to prepare lysates using RIPA or SDS-PAGE sample buffer to adequately release p64 from the nucleoli. If you are preparing samples using lysis buffer, you may sonicate your samples to release p64.
To detect p64 (human c-myc) expression, you may use any of the Anti-V5 Antibodies or the Anti-myc Antibodies available from Invitrogen.
Note: The c-myc gene encodes a protein with an expected molecular weight of 48 kDa, however, the native protein actually runs at a range of 55–64 kDa on an SDS-PAGE gel.
