Anti v5 antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Anti-V5 antibody is a laboratory reagent commonly used in various research applications. It is a monoclonal antibody that specifically binds to the V5 epitope, a small protein tag that can be engineered into recombinant proteins. The Anti-V5 antibody can be used for the detection, purification, or localization of V5-tagged proteins in experimental settings.

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189 protocols using anti v5 antibody

Immunofluorescence Staining of V5-Tagged Proteins

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For S2 cells, after 36-hours transfection, cells cultured on glass slides were fixed with 4% (w/v) paraformaldehyde in PBS for 20 min. Cells were washed three times with PBS and then permeabilized with 0.2% Triton X-100 for 15 min. After blocking with 5% (w/v) bovine serum albumin (BSA) in PBS for 1 hour, primary antibody incubation with anti-V5 antibody (1:500, Invitrogen, Cat. # 460705) in PBS was carried out overnight at 4 °C. Following three washes in PBS, cells were incubated with Alexa Fluor 488-labeled secondary antibody (1:500, Invitrogen, Cat. # A11029) and Hoechst 33342 (1:10000, Bio-Rad, Cat. # 151304) for 1 hour.
For Drosophila, ovaries from 14-day flies were dissected in Grace’s Insect Medium (Life) and then fixed with 4% (w/v) paraformaldehyde in PBS for 20 min. Ovaries were incubated with anti-V5 antibody (Invitrogen) in PBS containing 0.3% Triton X-100 and 0.5% horse serum overnight. To detect the distribution of biotinylated proteins and expression of ligase, ovaries were incubated with Alexa Fluor 488-labeled secondary antibody (1:500, Thermo Fisher) and Streptavidin-Cy3 (1:300, Jackson, Cat. # 016-160-084) containing Hoechst 33342 (Bio-Rad) overnight before confocal imaging.

Zhang B., Zhang Y, & Liu J.L. (2021). Highly effective proximate labeling in Drosophila. G3: Genes|Genomes|Genetics, 11(5), jkab077.

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Modulating GAPDH and ENO1 Expression in Cells

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Cells were transfected with 100 nM validated commercially available Silencer™ GAPDH siRNA (Catalog #:AM4605, Thermo Fisher Scientific Inc.) to suppress GAPDH expression and with 100 nM validated commercially available Stealth ENO1 siRNA (Catalog #:HSS103243, Thermo Fisher Scientific Inc.) to suppress ENO1 expression using the Neon™ transfection system (Thermo Fisher Scientific Inc.). To overexpress ENO1, cells were transfected with previously prepared pcDNA™3.1D-ENO1-V5-His-TOPO^® (Thermo Fisher Scientific Inc.; ENO1-V5 expression vector) using Lipofectamine^® LTX reagent and Plus™ Reagent (Thermo Fisher Scientific Inc.). To prepare the untagged ENO1 expression vector, the coding region of human ENO1 was cloned into pEBMulti-Neo (Wako Pure Chemical Industries, Ltd.). All experiments were performed in accordance with manufacturer’s instructions. The expression level of each protein was determined by western immunoblotting using anti-GAPDH antibody (Merck KGaA), anti-ENO1 antibody (Santa Cruz Biotechnology, Inc.), anti-V5 antibody (Thermo Fisher Scientific Inc.), anti-actin antibody (Wako Pure Chemical industries, Ltd.) or HIV-1-positive plasma (a gift from Dr. Matsushita, Kumamoto University). The cytotoxicity induced by siRNA treatment was evaluated by trypan blue dye exclusion assay [24 (link)].

Kishimoto N., Yamamoto K., Iga N., Kirihara C., Abe T., Takamune N, & Misumi S. (2020). Alpha-enolase in viral target cells suppresses the human immunodeficiency virus type 1 integration. Retrovirology, 17, 31.

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Biotinylation and Immunofluorescence Imaging

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Isolated cardiomyocytes were first exposed to biotin-phenol and H₂O₂ as described above. After quenching, the cells were fixed for 15 minutes in 4% paraformaldehyde, washed with glycine/PBS (phosphate-buffered saline) twice, treated with PBST (0.1% Triton X-100 (v/v) in PBS) for 5 minutes, and blocked with 3% BSA (w/v) in PBS for 1 hour. Indirect immunofluorescence was performed using dilutions of 1:500 of anti-V5 antibody (Thermofisher, R960-25) and 1:200 of Alexa594-labeled goat-anti-mouse antibody (Thermofisher, A11032, Lot#2069816), and 1:800 of streptavidin-Alexa Fluor 488 conjugate (Thermofisher, S32354, Lot# 1719656). Images were acquired using a confocal microscopy.

Liu G., Papa A., Katchman A.N., Zakharov S.I., Roybal D., Hennessey J., Kushner J., Yang L., Chen B.X., Kushnir A., Dangas K., Gygi S.P., Pitt G.S., Colecraft H.M., Ben-Johny M., Kalocsay M, & Marx S.O. (2020). Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics. Nature, 577(7792), 695-700.

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Antibody Screening for Human GPCRs

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mAbs reactive to ectodomain of human GPCRs were purchased from R&D system (MAB197, MAB330, MAB331, MAB190, MAB6594, MAB4139, MAB099, MAB4224, MAB8396, MAB42273, MAB150, MAB179, MAB8276, MAB2016, MAB8561, MAB145, MAB1567, MAB699, and MAB9305) except for P2RY13 which was from ProMab (30487). Anti-HSV-1 gD antibody DL6 was purchased from Santa Cruz Biotechnology (sc-21719) and ascities fluid for DL6 was a kind gift from David Johnson (OHSU). Anti-V5 antibody was purchased from Thermo Fisher Scientific (R960-25). Cy3-conjugated anti-mouse IgG was purchased from Jackson ImmunoResearch.

Syu G.D., Wang S.C., Ma G., Liu S., Pearce D., Prakash A., Henson B., Weng L.C., Ghosh D., Ramos P., Eichinger D., Pino I., Dong X., Xiao J., Wang S., Tao N., Kim K.S., Desai P.J, & Zhu H. (2019). Development and application of a high-content virion display human GPCR array. Nature Communications, 10, 1997.

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Flow Cytometry Analysis of TMPRSS2 Expression

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HEK293 cells transfected with plasmids expressing TMPRSS2 proteins were detached from culture plates with 0.02% (w/v) EDTA and incubated with an anti-V5 antibody (Thermo Fisher Scientific; catalog no.: R96025, 1:500 dilution) followed with an Alexa Fluor 488-labeled secondary antibody (Invitrogen; catalog no.: A21202, 1:500 dilution) or an anti-TMPRSS2 antibody (Abcam; catalog no.: ab280567, 1:200 dilution) followed with an Alexa Fluor 647-labeled secondary antibody (Yeasen; catalog no.: 33113ES60, 1:500 dilution). After washing with PBS, the cells were examined by flow cytometry (Gallios, Beckman Coulter). Pyridinium iodide (Sigma; catalog no.: P8080, 1:1000 dilution) was used for life cell gating. Data were analyzed with Kaluza Analysis Software (Beckman Coulter).

Zhang Y., Sun S., Du C., Hu K., Zhang C., Liu M., Wu Q, & Dong N. (2022). Transmembrane serine protease TMPRSS2 implicated in SARS-CoV-2 infection is autoactivated intracellularly and requires N-glycosylation for regulation. The Journal of Biological Chemistry, 298(12), 102643.

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Quantitative Analysis of PD-L1 Turnover

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V5-tagged PD-L1 transduced CMTM6 overexpressing A375 cells, and
V5-tagged PD-L1 transduced CMTM6 knockout A375 cells were cultured in
methionine- and cysteine-free medium for 1h at 37°C. Cells were then
pulse labeled with 0.5 mCi/ml [³⁵S]Cys/[³⁵S]Met
(PerkinElmer) for 1 hour. Cells were washed with PBS to remove residual
[³⁵S]Cys/[³⁵S]Met, and then cultured in regular medium
with extra ‘cold’ methionine and cysteine for 0, 1, 2, 3 and 6h.
Cell samples were lysed and used for immunoprecipitation with anti-V5 antibody
(ThermoFisher) immobilized on protein A or protein G coated beads
(ThermoFisher). Immunoprecipitates were either left untreated or treated with
EndoH or PNGaseF (New England Biolabs), according to the manufacturer’s
instructions.
Immunoprecipitates were run on NuPAGE 4-12% gels. Gels were treated with
1M NaSalicylate pH5.6 before drying, and then analysed on Fujifilm BAS-MP
phosphor imager screens. Quantification was performed using a Fujifilm FLA-3000
phosphorimager and AIDA image analyzer software. Gels were exposed to film using
intensifier screens at -80 C.

Mezzadra R., Sun C., Jae L.T., Gomez-Eerland R., de Vries E., Wu W., Logtenberg M.E., Slagter M., Rozeman E.A., Hofland I., Broeks A., Horlings H.M., Wessels L.F., Blank C.U., Xiao Y., Heck A.J., Borst J., Brummelkamp T.R, & Schumacher T.N. (2017). Identification of CMTM6 and CMTM4 as PD-L1 protein regulators. Nature, 549(7670), 106-110.

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Characterization of Recombinant EHDV VP7 Protein

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Recombinant VP7 purified from Sf9 cell pellet and supernatant were checked for purity by SDS-PAGE and characterized by Western blotting using the anti-VP7 mAb, clone 4G11 (IZSLER) raised against semipurified EHDV field strain serotype 7 obtained from Kimron Institute (Israel) and the anti-V5 antibody (Thermo Fisher Scientific, Cat#R961-25, RRID: AB_2556565) both conjugated with HRP. Uninfected Sf9 cells were used as negative control.
Briefly, the same quantity of purified protein derived from cell pellet and supernatant was denatured for 10 min at 70°C, as suggested by Thermo Fisher Scientific instructions, separated on NuPAGE Novex 4–12% Bis-Tris Gels (Thermo Fisher Scientific, #CatNP0321BOX) and stained with Biosafe Coomassie G-250 stain (Bio-Rad, #Cat 1610786).
Recombinant VP7 samples fractionated by electrophoresis were also transferred onto nitrocellulose membranes and tested for immunoreaction, incubating the membranes with the anti-VP7 mAb clone 4G11 (IZSLER) and the anti-V5 antibody at 1 : 30000 dilution and 1 : 10000 dilution, respectively, both conjugated with HRP, at 4°C overnight. After rinsing membranes with PBST, the Amersham ECL Select Western Blotting Detection Reagent (Cytiva, Cat# RPN2235) was used to acquire images of EHDV recVP7.

Serroni A., Ulisse S., Iorio M., Laguardia C., Testa L., Armillotta G., Caporale M., Salini R., Lelli D., Wernery U., Raghavan R., Mercante M.T, & Di Ventura M. (2022). Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels. Journal of Tropical Medicine, 2022, 5210771.

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Biotinylation and Immunofluorescence Imaging

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Immunoprecipitation of S1R-Apex Complex

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Lysates were prepared from HeLa cells stably expressiong S1R-Apex using a 0.2% CHAPS buffer (0.2% CHAPS, 50 mM Tris-Cl pH 7.5, 150 mM NaCl, and 1 mM EDTA). 1000ug of total protein was used in each immunoprecipitation experiment. Anti-HA antibody (Santa Cruz Biotechnology) was used as a control and anti-V5 antibody (ThermoFisher) was used to precipitate S1R-Apex. The respective antibodies were incubated with the lysates for 1 h at 4°C. Next, the antibody complexes were pufiried using protein-A agarose beads (Pierce) at 4°C for 1 h. The beads were washed four times with 800 ul of 0.2% CHAPS buffer. The bounds proteins were eluted from the beads by boiling in Laemmli buffer and analyzed by Western blotting using the indicated antibodies.

Zhao J., Veeranan-Karmegam R., Baker F.C., Mysona B.A., Bagchi P., Liu Y., Smith S.B., Gonsalvez G.B, & Bollinger K.E. (2023). Defining the ligand-dependent proximatome of the sigma 1 receptor. Frontiers in Cell and Developmental Biology, 11, 1045759.

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Overexpression of Rat BDNF in HEK293 Cells

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Rat Bdnf coding sequence was cloned into pcDNA3.1/V5-His-TOPO expression vector (Life Technologies) where the CMV promoter was replaced with EF1α promoter. HEK293 cells were grown in Minimum Essential Medium (MEM, Corning, cat. no 10-010-CV) supplemented with 10% fetal bovine serum (PAN Biotech), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). The cells were transfected on 12-well plate with PEI (Sigma) with 1.5 μg DNA per well using 1:2 DNA:PEI ratio. Cells were lysed 24 h post transfection directly into 1x Laemmli buffer (containing 5% β-mercaptoethanol) and proteins were subjected to SDS-PAGE and Western blot using anti-V5 antibody (Thermo Fisher Scientific, #R960-25, 1:5000) or anti-BDNF 3C11 antibody.

Esvald E.E., Tuvikene J., Kiir C.S., Avarlaid A., Tamberg L., Sirp A., Shubina A., Cabrera-Cabrera F., Pihlak A., Koppel I., Palm K, & Timmusk T. (2023). Revisiting the expression of BDNF and its receptors in mammalian development. Frontiers in Molecular Neuroscience, 16, 1182499.

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