V5 tag

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The V5 tag is a short peptide sequence that can be used to label and detect recombinant proteins. It provides a consistent and reliable way to identify and quantify target proteins in various experimental settings.

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Lab products found in correlation

Flag tag (flag), by Merck Group (11 mentions) β actin, by Merck Group (6 mentions) β actin, by Santa Cruz Biotechnology (4 mentions) Myc tag, by Cell Signaling Technology (3 mentions) Vinculin, by Merck Group (3 mentions) Abc kit, by Vector Laboratories (2 mentions) Hrp conjugated secondary antibody, by Vector Laboratories (2 mentions) V5 hrp, by Thermo Fisher Scientific (2 mentions) C myc, by Santa Cruz Biotechnology (2 mentions) Ha tag, by Merck Group (2 mentions) Gapdh, by Thermo Fisher Scientific (2 mentions) Gapdh, by Merck Group (2 mentions) β actin, by Thermo Fisher Scientific (2 mentions) Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions) Skim milk, by Merck Group (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Nupage lds sample buffer 4x, by Thermo Fisher Scientific (2 mentions) Mief2, by Atlas Antibodies (2 mentions) Flag m2, by Merck Group (2 mentions) Anti mouse igg hrp, by Cell Signaling Technology (2 mentions)

Close spelling variants of this product

Anti-V5 tag (21 citations) Anti-V5 tag antibody (16 citations) V5-tag antibody (12 citations) V5 Tag mouse monoclonal antibody (8 citations) Mouse anti-V5 tag monoclonal antibody (6 citations) Mouse anti-V5 tag (6 citations) V5 Tag Monoclonal Antibody (5 citations) PMT-Bip-V5-His tag vector (5 citations) V5 epitope tag antibody (5 citations) MAb anti-V5 epitope tag (4 citations)

51 protocols using v5 tag

Antibody generation and immunoblotting protocol

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Affinity-purified rabbit polyclonal antibody against AMAP1 was as described previously.^{21 (link)} Rabbit polyclonal antibody against EPB41L5 was raised against a GST-fused peptide corresponding to amino acids (aa) 541–733 of EPB41L5. The resulting serum was adsorbed with GST and then affinity-purified using the antigen peptide, before use. Other antibodies were purchased from commercial sources: mouse monoclonal antibodies against p53 (#2524, Cell Signaling Technology, Beverley, CA, USA), V5-tag (#R960-25, Invitrogen), HA-tag (#MMS-101R, Biolegend, San Diego, CA, USA), E-cadherin (#610182, BD Biosciences), and β-actin (#A5441, Sigma-Aldrich); and rabbit polyclonal antibodies against Smad2 (#3103), Ser465/467-phosphorylated Smad2 (#3101), ZEB1 (#3396), Snail (#3879), and Slug (#9585) (all from Cell Signaling Technology). Donkey antibodies against rabbit (#711-036-152) and mouse immunoglobulins G (#711-036-151), each conjugated with horseradish peroxidase, were from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Immunoblotting analysis was performed by use of ECL kit (GE Healthcare Life Sciences, Piscataway, NJ, USA), as described previously.^{20 (link), 21 (link), 24 (link), 25 (link)}

Hashimoto A., Hashimoto S., Sugino H., Yoshikawa A., Onodera Y., Handa H., Oikawa T, & Sabe H. (2016). ZEB1 induces EPB41L5 in the cancer mesenchymal program that drives ARF6-based invasion, metastasis and drug resistance. Oncogenesis, 5(9), e259-.

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Targeted RNA-seq of RBP-ADAR Fusion

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The RBP of interest was cloned upstream of the Drosophila ADAR catalytic domain (the whole C terminus downstream of the 2^nd dsRBD was used, starting Y268 to terminal E669 of AHN59262.1) with minimal linker region into a pMT-A vector (Invitrogen, V4120-20, also harboring a blasticidin resistance gene). Stable S2 cell lines were made by transfecting with the pMT-RBP-ADARcd-V5-Blasticidin plasmid followed by subsequent blasticidin resistance selection. Fusion protein expression was induced by introduction of copper sulphate for 24 hours, prior to harvesting protein and RNA. S2 cell expression of all fusion proteins was assayed by western blot for the V5 tag (Invitrogen, 46-1157). Nascent RNA was extracted from S2 cells, according to Khodor et al. (2011) (link) and depleted of ribosomal RNA according to Pennington et al. (2013) (link) and mRNA (two rounds of pA depletion using Invitrogen Dynabeads Oligo dT, according to manufacturer's protocol).
Fluorescently labelled neurons were isolated from dissected, triturated fly brains by manual sorting using a glass micro-pipette, as described in Abruzzi et al. (2015) (link). RNA sequencing libraries from S2 cells were constructed using the standard Illumina Truseq kit protocol. RNA-sequencing libraries from manually sorted neurons were made as described in Abruzzi et al. (2015) (link).

McMahon A.C., Rahman R., Jin H., Shen J.L., Fieldsend A., Luo W, & Rosbash M. (2016). Hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins. Cell, 165(3), 742-753.

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Western Blot Protocol for Protein Detection

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Proteins separated by SDS-PAGE were electrophoretically transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore), which were then blocked at room temperature with 5% nonfat dry milk powder and 0.2% Tween-20 dissolved in phosphate-buffered saline (PBS) containing 154 mM NaCl and 10 mM Na₂PO₄ (pH 7.4). The membranes were incubated for 2 hr at room temperature or overnight at 4°C with primary antibodies at a 1:10,000 dilution in blocking solution, and washed with PBS containing 0.2% Tween-20. The membranes were then incubated for 2 hr with horseradish peroxidase (HRP)-decorated goat anti-rabbit secondary antibodies (SeraCare, Cat. No. 5220–0341) at 1:10,000 dilution in blocking solution, followed by washes with PBS containing 0.2% Tween-20. Chemiluminescence was generated using the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher) and captured with X-ray film. Primary polyclonal antibodies included those against AtSUMO1 [20 (link)], histone H3 (Abcam; ab1791), α and γ zeins [41 (link)], the T7 Tag polyclonal antibody coupled to HRP (Invtrogen; Cat. No. PA1-33133), and the V5 tag (Invitrogen; Cat. No. PA1-993).

Zhang J., Augustine R.C., Suzuki M., Feng J., Char S.N., Yang B., McCarty D.R, & Vierstra R.D. (2021). The SUMO ligase MMS21 profoundly influences maize development through its impact on genome activity and stability. PLoS Genetics, 17(10), e1009830.

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Cloning and Tagging of RARA and RXRB

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Wild-type RARA and RXRB entry clones were obtained from the hORFeome v8.1^{71 (link)} collection. Gateway LR reactions were used to transfer bait RARA wild-type, p.Pro375Leu, and p.Arg83His into a pQXIP (ClonTech, 631516) vector modified to include a Gateway cassette featuring a C-terminal 3×FLAG. Prey RXRB was transferred into pcDNA-DEST40 which includes a V5 tag (Invitrogen, 12274–015) also using Gateway LR reactions.

Chen S., Fragoza R., Klei L., Liu Y., Wang J., Roeder K., Devlin B, & Yu H. (2018). An interactome perturbation framework prioritizes damaging missense mutations for developmental disorders. Nature genetics, 50(7), 1032-1040.

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Wnt Signaling Pathway Modulation

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pCDNA-V5-FZD5 was a kind gift from Dr. Hans Clevers. TCF4-DN in pLX303 was a gift from Dr. William Hahn (Addgene plasmid # 42592). Two distinct DsiRNA-DDB2, two DsiRNA-EZH2 and non-targeting DsiRNA-Control were purchased from IDT DNA pre-designed library. The sequences are listed in supplementary table S1. ON-TARGET plus PAF siRNA smartpool and non-targeting siRNA smartpool were purchased from Dharmacon. Antibodies used in this study include DDB2 (Western Blotting: 5416, Cell Signaling; Chromatin Immunoprecipitation: sc-25368, Santa Cruz; co-Immunoprecipitation: ab181136, Abcam), EZH2 (Western Blotting: 5246, Cell Signaling; Chromatin Immunoprecipitation: 39875, Active Motif), β-catenin (8480, Cell Signaling), RNF43 (orb140091, Biorbyt), EED (sc-28701, Santa Cruz), SUZ12 (3737, Cell Signaling), TCF4 (2569, Cell Signaling), V5 tag (R960-25, Invitrogen), T7 tag (69522, EMD Millipore), α-Tubulin (T9026, Sigma-Aldrich) and LRP5/6 (bs-2905R, BIOSS). HRP-conjugated secondary antibodies were purchased from BioRad.

Huang S., Fantini D., Merrill B.J., Bagchi S., Guzman G, & Raychaudhuri P. (2017). DDB2 is a Novel Regulator of Wnt-Signaling in Colon Cancer. Cancer research, 77(23), 6562-6575.

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Immunohistochemical Staining of Brain Sections

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IHC was performed as previously reported [32 (link),35 (link)]. Briefly, the mice were transcardially perfused with 10% neutralized formalin and the brains were removed and immersed in 25% sucrose solution, cut into five series of 30 μm sections and stored in cryoprotectant at 20 °C until processed for IHC. Brain sections were rinsed in PBS, blocked in 3% normal donkey serum and 0.3% Triton X-100 in PBS for 30 min at room temperature. Sections were then incubated with primary antibodies against GFP (Aves Labs), mCherry (Clontech), c-Fos (CalBioChem), or V5 tag (Invitrogen) overnight at 4 °C, and then washed and incubated with Cy2-, Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) for fluorescent visualization or with HRP-conjugated secondary antibody for chromogenic staining using an ABC kit as per the manufacturer’s instructions (Vector Laboratories).

Singh U., Saito K., Khan M.Z., Jiang J., Toth B.A., Rodeghiero S.R., Dickey J.E., Deng Y., Deng G., Kim Y.C, & Cui H. (2023). Collateralizing ventral subiculum melanocortin 4 receptor circuits regulate energy balance and food motivation. Physiology & behavior, 262, 114105.

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Western Blot Analysis of V5-Tagged Proteins

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Cell pellets were collected and resuspended in lysis buffer (600mM Nacl, 1% Triton X-100 in PBS, 1 X protease inhibitor). 30 εg of total protein lysates of each sample was separated by electrophoresis in 10 % SDS-PAGE gels and transferred onto 0.45 εm PVDF transfer membranes using a Semi-Dry transfer cell (Trans-Blot SD, Bio-Rad). Membranes were blocked for 1 h at room temperature using blocking buffer (5 % nonfat milk in TBST) and incubated with antibodies against V5 Tag, or V5-HRP (Invitrogen), or β-actin (Santa Cruz). Next, membranes were washed three times using TBST, 5 min each. With the exception for V5-HRP in Western blot assays, HRP-conjugated anti-rabbit IgG or anti-mouse IgG was used as secondary antibody (Invitrogen) followed by a 45 minutes incubation and washing as described above. Chemiluminescence signal was developed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Gao P., Xia J.H., Sipeky C., Dong X.M., Zhang Q., Yang Y., Zhang P., Cruz S.P., Zhang K., Zhu J., Lee H.M., Suleman S., Giannareas N., Liu S., Tammela T.L., Auvinen A., Wang X., Huang Q., Wang L., Manninen A., Vaarala M.H., Wang L., Schleutker J, & Wei G.H. (2018). Biology and clinical implications of the 19q13 aggressive prostate cancer susceptibility locus. Cell, 174(3), 576-589.e18.

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Cloning and Tagging of Protein Constructs

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Wild-type MLH1, HSPA8, and BRIP1 entry clones are from the human ORFeome v8.1 collection [61] (link). Using Gateway LR reactions, wild-type MLH1, mutant MLH1 (I107R), and GFP were transferred into the pMSCV-N-FLAG-HA-PURO vector [65] (link); HSPA8 and BRIP1 were transferred into the pcDNA-DEST40 vector that contains a C-terminal V5 tag (Invitrogen 12274-015).

Wei X., Das J., Fragoza R., Liang J., Bastos de Oliveira F.M., Lee H.R., Wang X., Mort M., Stenson P.D., Cooper D.N., Lipkin S.M., Smolka M.B, & Yu H. (2014). A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations. PLoS Genetics, 10(12), e1004819.

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Antibody Characterization for Cell Biology

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Antibodies were purchased from the following commercial sources: EGFR C-terminal (Invitrogen; Cat # PA1–1110; 1:200), EGFR N-terminal (E234; Abcam; Cat # ab32198; 1:1000), V5-tag (Invitrogen; Cat # 46–0705; 1:5000), c-Myc (9E10, Santa Cruz Biotechnologies; Cat # sc-50; 1:500), EEA1 (Thermo Fisher; Cat # PA5–17228; 1:500), LAMP1 (H4A3; Developmental Studies Hybridoma Bank; Cat # H4A3; 1:500), Rab11 (Millipore; Cat # 2413S; 1:1000), Rab 7 (Invitrogen; Cat #D95F2; 1:1000), Transferrin Receptor (TfR, Abcam; Cat # ab190640; 1:1000).

Gireud-Goss M., Reyes S., Wilson M., Farley M., Memarzadeh K., Srinivasan S., Sirisaengtaksin N., Yamashita S., Waxham M.N., Tsunoda S., Lang F.F, & Bean A.J. (2018). Distinct mechanisms enable inward or outward budding from late endosomes/multivesicular bodies. Experimental cell research, 372(1), 1-15.

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Curdlan-Induced NFATc2 Translocation Assay

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D1 cells were seeded into 8 well μ-slides (Ibidi) and rested for 2 h before stimulation with curdlan (10 μg/ml) for various times prior to fixation in 2% paraformaldehyde (PFA). The cells were then re-suspended in permeabilization buffer (0.1% saponin in D-PBS containing 5-mg/ml bovine serum albumin and 0.2% gelatin), immunolabeled with antibodies against NFATc2 (Thermo Scientific) or V5 tag (Invitrogen) and finally counter-stained with 4', 6-diamidino-2-phenylindole (DAPI) and anti-Alexa488 antibody (Life technologies).

Yu H.B., Yurieva M., Balachander A., Foo I., Leong X., Zelante T., Zolezzi F., Poidinger M, & Ricciardi-Castagnoli P. (2014). NFATc2 mediates epigenetic modification of dendritic cell cytokine and chemokine responses to dectin-1 stimulation. Nucleic Acids Research, 43(2), 836-847.

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