Mammalian vector for expressing cterminally v5 and 6xhistagged proteins. Immunohistochemical detection of transgene expression in. Mammalian v5 tag vector set plasmid vectors for molecular cloning. All pef or pub vectors contain a strong promoter for highlevel expression in. B sox2 gene tagged independently with four different epitope tags in mouse ns bl6 cells, bulk population icc images for each epitope tag are shown. Tag 5 are a series of epitope tagging mammalian expression vectors.
The v5 tag is generally used with all 14 amino acids gkpipnpllgldst, but it may also be used with a shorter 9amino acid ipnpllgld sequence. Briefly, the 3x v5 epitope was polymerase chain reaction pcramplified from the plasmid zm474 24 using. Cterminal v5 epitope tag for efficient detection of recombinant proteins with an antiv5 antibody. In addition, the vector includes the following features. Evaluation of epitope tag expression in cell extracts.
Welcome to vector database vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Escs were cotransfected with a grna designed near the stop codon and a repair template singlestranded oligo or targeting vector containing an epitope tag flag, v5 or ha. Representative bulk population v5 icc images for each tf are shown. Endogenous epitopetagging of tet1, tet2 and tet3 identifies. Use the list below to choose the v5 epitope tag antibody which is most appropriate for your research. Epitope tagging technology provides you a powerful means to functionally analyze your protein without the need for an antibody specific to each new protein under study.
The v5 epitope tag is derived from a small epitope pk present on the p and v proteins of the paramyxovirus of simian virus sv5. More than 250 well selected v5 epitope tag antibodies available. Internal epitope tagging informed by relative lack of. For epitope tag affinity matrices, prepare an elution buffer with epitope tag peptide at 400 ugml in 50 mm trishcl ph 7. Antiau5 epitope tag affinity matrix previously covance. Our pcmv3tag vector series is an advanced epitope tagging system in which three copies of either the flag or cmyc tags are added to your protein. Tags function as universal epitopes, easily detected by commercially available antibodies, and generally do not compromise the native structure or function of the protein. The v5 epitope tag serves as a linker between dam and your favorite protein. The epitope is, of course, the portion of the antigen protein to which the antibody binds.
To construct a recombinant plasmid containing the sequence of the v5 epitope, fusion. Epitope tags such as v5, cmyc, and ha are biological structures, usually proteins, that are genetically engineered onto either the n or c terminus of a recombinant protein. If you wish to include the v5 epitope tag, your gene in the entry clone should not contain a stop codon. Epitope tagging is a recombinant dna method by which a protein encoded by a cloned gene is made immunoreactive to a known antibody. Christopher newgards lab contains the insert v5 epitope tag open and is published in nucleic acids res.
These are usually derived from viral genes, which explain their high immunoreactivity. Expanding the repertoire of plasmids for pcrmediated epitope. The v5 tag is usually used with all 14 amino acids gkpipnpllgldst, although it has also been used with a shorter 9 amino acid sequence ipnpllgld. Flow cytometry v5 epitope tag antibody mab8926 hek293 human embryonic kidney cell line transfected with v5 tagged proteins. C mouse bl6 were transfected with rnps and v5encoding ultramers for v5 epitope tagging of indicated genes.
This section discusses the design of pcr primers for epitope tagging. Expanding the repertoire of plasmids for pcrmediated. The topo box contains linearized and topoisomerase iactivated pcdna3. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. This is a free resource for the scientific community that is compiled by addgene. Nov 28, 2016 testing for tag integration was done by performing pcr using primer pairs tcf21f1 v5 r1, tcf21r1 v5 f1, tbx18f1 v5 r1 or tbx18r2 v5 f1. For chipseq studies, ampr was tagged at the carboxyterminus with a 3x v5 epitope tag. V5 epitope tag antibody detection of 200, 100, or 50 ng of e.
Home resources plasmid files insect cell vectors piz v5his pizv5his compact insect cell vector with a zeocin resistance marker, for constitutive expression of proteins with an optional cterminal v56xhis tag. Our results show that all the examined small epitope tags could be detected by anti tag antibodies both in cell extracts as well as in vivo, although to varying degrees depending on the tag and antibody used. The v5 tag is derived from a small epitope pk found on the p and v proteins of the paramyxovirus of the simian virus 5 sv5 family. Allow sequence of interest to be fused to nterminal double v5 epitope tag. Our v5 epitope tag antibodies can be used in a variety of model species. This short peptide sequence was chosen because highaffinity antibodies can be reliably produced in many different species southern et al. Epitope tags include alfa tag, v5 tag, myc tag, ha tag, spot tag, t7 tag and ne tag. V5 tag antibodies provide a dependable method for the detection and purification of.
For complete details, download the datasheet via the get pdf button. Elution by ph or chaotropic salts is also possible. C mouse bl6 were transfected with rnps and v5 encoding ultramers for v5 epitope tagging of indicated genes. B, c immunofluorescence for flag tet1, red, v5 tet2, yellow, and ha tet3, magenta in tet tag tag escs cultured in serumlif b or 2ilif c. This is a free resource for the scientific community that is compiled by addgene this page is informational only this vector is not available from addgene please contact the manufacturer for further details. Hek293t cells were transduced with lentiviral vectors encoding different tagegfp fusion proteins or egfp alone table 1 and fig. Epitope tags and the v5 tag antibody novus biologicals. Antiflag antibody purchased from sigma for resale under license from sigmaaldrich co. The amount of vector was normalized for expression based on functional titers transducing units. Briefly, the 3xv5 epitope was polymerase chain reaction pcramplified from the.
All pcdna vectors contain a strong promoter for highlevel expression in mammalian cells, a choice of selection marker for generating stable cell lines, and an epitope tag for easy detection with a monoclonal antibody and rapid purification on nickelchelating resin. Invitrogens mammalian expression vectors product list filtered by, v5 epitope tag. A gatewaybased vector system for adaptive expression. The recognized v5 epitope represents 95 gkpipnpllgldst 108 of rna polymerase. Epitope tags and the v5 tag antibody antibody news. Testing for tag integration was done by performing pcr using primer pairs tcf21f1v5r1, tcf21r1v5f1, tbx18f1v5r1 or tbx18r2v5f1. Working with the v5 tag antibody the v5 tag antibody identifies the v5 epitope tag, enabling proteins of interest to be analyzed and studied via elisa, western blot and other immunochemical methods. Epitope tags include alfatag, v5tag, myctag, hatag, spottag, t7tag and netag. Construction and biological characterisation of recombinant porcine circovirus type 2 expressing the v5 epitope tag article in virus research 1612. The 3xty1 tag worked very well even when the protein of interest was expressed at pretty low levels. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. The ha tag is derived from the hamolecule corresponding to amino acids 98 106 has been extensively used as a general epitope tag in expression vectors. In addition, the gene should be in frame with the v5 epitope tag after recombination. In the paul vector system, the ha epitope and streptagiii can be used for single tagfusions of proteins of interest for detection ha or.
Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. With this technique, expression of the fusion protein is monitored using a tagspecific. Apr 11, 2018 b sox2 gene tagged independently with four different epitope tags in mouse ns bl6 cells, bulk population icc images for each epitope tag are shown. The v5 tag antibody recognizes the artificially engineered v5 epitope, which is formed of amino acid residues 95108 of the simian virus 5 rna polymerase alpha subunit. In some instances, v5 f11 was used instead of v5 f1 for. We offer an extensive range of high performance great value epitope tag antibodies and antireporter protein antibodies. Tags function as universal epitopes, easily detected by commercially available antibodies, and generally do not compromise the native structure or. Lentiviral gateway destination vector with a blasticidin resistance marker and a v5 epitope tag, for strong expression and improved plasmid yield and viral titer.
Addition of an epitope tag sequence university of ottawa. B, c immunofluorescence for flag tet1, red, v5 tet2, yellow, and ha tet3, magenta in tet tagtag escs cultured in serumlif b or 2ilif c. I also have word from another colleague that the biotag works well for chip but that requires introducing a second plasmid into your cells. We at novus biologicals are one of the leading suppliers of epitope tag reagents, wit. Strong cmv promoter for highlevel, constitutive expression. For chipseq studies, ampr was tagged at the carboxyterminus with a 3xv5 epitope tag.
This sequence is from the cterminal sequence of the p and v proteins of simian virus 5. If, however, the protein function will be disrupted by a terminal tag, this vector also allows. In some instances, v5f11 was used instead of v5f1 for. If you do not wish to include the v5 epitope tag, make sure that your gene padpldest padpldest. V5 tag antibodies provide a dependable method for the detection and purification of tagged target proteins without a.
Epitope tagging is widely used in proteomics and genetic studies. May 16, 2018 epitope tagging is accomplished by fusion of the target protein with the tag of choice. Using the presented protocol, v5 anti v5 and haha11 tag antibody combinations provided the most sensitive detection in brain tissue. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. An efficient and scalable pipeline for epitope tagging in. This is accomplished by insertion of the target gene into a host cellspecific expression vector that also encodes the epitope tag.
Proteins were transferred to a pvdf membrane and blocked at 4c overnight. Our range includes v5 tag antibodies, his6 tag antibodies, cmyc tag antibodies and dykddddk tag monoclonal antibodies. Immunohistochemical detection of transgene expression in the. One more important thing to consider when using epitope tags for chip is expression level. Epitope tag antibodies our monoclonal antibodies against epitope tags are provided purified at a concentration of 1 mg ml. The v5 epitope tag is derived from a small epitope pk present on the p and v proteins of the paramyxovirus of simian virus 5 sv5. When transfected as a conventional plasmid, the strong viral promoter drives high expression of the damfusion proteins. For a free vector file viewer, try ape or snapgene viewer.