pcDNA6.2/cTC-Tag-DEST

pcDNA6.2/cTC-Tag-DEST

pcDNA6.2/cTC-Tag-DEST载体基本信息

pcDNA6.2/cTC-Tag-DEST载体质粒图谱和多克隆位点信息

pcDNA6.2/cTC-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 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

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.

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.

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.

载体序列

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT

AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA

ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG

ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC

ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG

CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC

ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC

AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA

TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC

AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA

TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG

CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA

CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTGAGC

ATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAG

ATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATT

AGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCG

AGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCC

AATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCA

GCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCAC

ATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATAT

GGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGA

ATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTG

GCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCA

GTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATAC

GCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTC

GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCG

GCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGA

TATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG

ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAAT

GAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGT

TTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCT

ATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACG

GATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTG

CATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGG

AAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAAT

ATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTT

ACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTT

TCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATGCTGTTAACGGGAAGCCTATCCCTAACCCTCTCCTCG

GTCTCGATTCTACGCGTACCGGTGCTGGTGGCTGTTGTCCTGGCTGTTGCGGTGGCGGCTAGTAATGAGT

TTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAAT

AAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTG

GCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCC

CACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAG

ATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGT

GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCT

TCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGAT

TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC

CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT

GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATT

GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGG

TGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCA

GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC

CATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCAT

GGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGT

GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATC

TGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGA

ACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAA

CAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACT

GGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTG

CGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGG

ACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAG

CCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCC

GAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCG

GAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCA

CCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA

GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATAC

CGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT

CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA

CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT

GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA

GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG

CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCA

GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT

CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC

ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC

CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC

TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT

TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC

AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTT

GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT

CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC

CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC

AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT

GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC

GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGA

AGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT

CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG

TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA

GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA

TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTC

AACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT

ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA

GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT

TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG

ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC

TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG

AAAAGTGCCACCTGACGTC

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