Anti v5 tag antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Anti-V5 tag antibody is a recombinant monoclonal antibody that specifically binds to the V5 epitope tag. The V5 tag is a short peptide sequence commonly used to facilitate the detection and purification of recombinant proteins expressed in various systems. The Anti-V5 tag antibody can be used to detect and immunoprecipitate proteins containing the V5 tag.

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Lab products found in correlation

Anti v5 tag, by Thermo Fisher Scientific (2 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Sangon (2 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Tgx stain free fastcast acrylamide kit, by Bio-Rad (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions) Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Chemidoc mp system, by Bio-Rad (1 mentions) Anti his hrp antibody, by Santa Cruz Biotechnology (1 mentions) Anti β catenin antibody, by BD (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Dm2500, by Leica (1 mentions) A11008, by Thermo Fisher Scientific (1 mentions) A21203, by Thermo Fisher Scientific (1 mentions) R960 25, by Thermo Fisher Scientific (1 mentions) Anti flag tag antibody, by Cell Signaling Technology (1 mentions) H 1500, by Vector Laboratories (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) P5726 5ml, by Merck Group (1 mentions) Mca465ga, by Bio-Rad (1 mentions)

Spelling variants of this product

Anti-V5 (240 citations) Anti-V5 antibody (189 citations) V5 tag (51 citations) V5 antibody (51 citations) Anti-V5 tag (21 citations) V5-tag antibody (12 citations) Mouse anti-V5 tag monoclonal antibody (6 citations) Anti-V5 Tag primary antibody (2 citations) Anti-V5 tag antibody (R960-25) (2 citations) Anti-V5-HRP antibody for tag detection (2 citations)

16 protocols using anti v5 tag antibody

Protein Extraction and Western Blot Analysis

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Protein extraction was performed with 100 μL RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF, Beyotime, Shanghai, China) by using the TGX Stain-Free FastCast Acrylamide Kit (Bio-Rad, 1610182, Hercules, CA, USA). The extracted proteins were separated by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a semidry transfer membrane. The membrane was sealed with a sealing solution (Bio-Rad, P0023B, Shanghai, China) at 25 °C for 2 h and then incubated with the following antibodies at 4 °C for 12 h: purified anti-Vg antibody (1:1000), anti-30Kc19 antibody (1:1000), anti-V5 tag antibody (1:1000, AB_2533339, Thermo Fisher, Waltham, MA, USA), and anti-α-tubulin antibody (1:5000, T0033, Affinity). After three times washing with Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST; pH 7.5), the membrane was incubated with HRP-conjugated anti-mouse IgG (1:5000, Bioworld Technology, St Louis Park, MN, USA) at 37 °C for 2 h. After washing the membrane again with TBST three times, an appropriate amount of ECL (Bio-Rad) coloring solution (1:1) was added, and the membrane was kept incubated in dark. Image Lab software was used to process and analyze the generated images.

Yuan Q., Sun X., Lu R., Qu Z., Ding X., Dai T., Qiu J., Tan Y., Zhu R., Pan Z., Xu S, & Sima Y. (2023). The LIM Domain Protein BmFHL2 Inhibits Egg Production in Female Silkworm, Bombyx mori. Cells, 12(3), 452.

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Western Blot Analysis of Protein Samples

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The SDS-PAGE and western blot were carried out according to prior methodology [36 (link)]. Briefly, Aag2 cells were lysed in RIPA buffer and 15–20 µg of each cell lysate or 4–6 µg of purified protein samples were resolved in SDS-PAGE gel and then transferred onto the nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were probed with the following primary antibodies: anti-His HRP antibody (Santa Cruz, Dallas, TX, USA, Cat. no. sc-8036-HRP, 1:5000), anti-actin HRP (C4) (Santa Cruz, Dallas, USA, Cat. no. sc-47778 HRP, 1:6000), anti-pADPr antibody (10H) (Santa Cruz, Dallas, USA, Cat. no. sc-56198, 1:3000 dilution), and anti-V5 tag antibody (Thermo Fisher Scientific, Waltham, MA, USA, Cat. no. R960-25, 1:5000) and anti-CHIKV E1 (in house raised in mice, 1:3000). The blots probed with anti-pADPr antibody, anti-CHIKV E1 sera, and anti-V5 tag antibody were incubated with anti-mice IgG HRP antibody (Novus Biologicals, Colorado, USA, Cat. no. NB7539, 1:6000 dilution), washed with PBST, and then visualized using the Bio-Rad ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA, USA) after brief exposure to a chemiluminescent substrate. The uncropped images are included in supplementary information Figure S1.

Kumar R., Mehta D., Nayak D, & Sunil S. (2023). Characterization of an Aedes ADP-Ribosylation Protein Domain and Role of Post-Translational Modification during Chikungunya Virus Infection. Pathogens, 12(5), 718.

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Protein Extraction and Western Blotting for β-Catenin

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Extraction of protein from HEK293T cells transfected with β-catenin expression constructs and luciferase reporter plasmids and western blotting were performed as previously described.^{23 (link)} Primary antibodies used in this study were anti-Myc tag 9E10 antibody (1:2000), anti-V5 tag antibody (1:2000, ThermoFisher Scientific, Cat #R960-25), anti-β-catenin antibody (1:1000, BD transduction laboratories, Cat#610153), and anti-β-actin antibody (1:2000, Sigma, Cat #A2228).

Kayumi S., Pérez-Jurado L.A., Palomares M., Rangu S., Sheppard S.E., Chung W.K., Kruer M.C., Kharbanda M., Amor D.J., McGillivray G., Cohen J.S., García-Miñaúr S., van Eyk C.L., Harper K., Jolly L.A., Webber D.L., Barnett C.P., Santos-Simarro F., Pacio-Míguez M., del Pozo A., Bakhtiari S., Deardorff M., Dubbs H.A., Izumi K., Grand K., Gray C., Mark P.R., Bhoj E.J., Li D., Ortiz-Gonzalez X.R., Keena B., Zackai E.H., Goldberg E.M., de Nanclares G.P., Pereda A., Llano-Rivas I., Arroyo I., Fernández-Cuesta M.Á., Thauvin-Robinet C., Faivre L., Garde A., Mazel B., Bruel A.L., Tress M.L., Brilstra E., Fine A.S., Crompton K.E., Stegmann A.P., Sinnema M., Stevens S.C., Nicolai J., Lesca G., Lion-François L., Haye D., Chatron N., Piton A., Nizon M., Cogne B., Srivastava S., Bassetti J., Muss C., Gripp K.W., Procopio R.A., Millan F., Morrow M.M., Assaf M., Moreno-De-Luca A., Joss S., Hamilton M.J., Bertoli M., Foulds N., McKee S., MacLennan A.H., Gecz J, & Corbett M.A. (2022). Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants. Genetics in medicine : official journal of the American College of Medical Genetics, 24(11), 2351-2366.

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Visualizing Recombinant PCSK6 and Corin in Cardiomyocytes

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To visualize recombinant PCSK6 and corin proteins in cardiomyocytes, cultured HL-1 cells were transfected with plasmids expressing human PCSK6 and WT or sWT corin. After 24 h, the cells were fixed with 3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, and incubated with an anti-flag tag antibody (Cell Signaling, 14793) for recombinant PCSK6 or an anti-V5 tag antibody (ThermoFisher, R96025) for recombinant corin proteins at 4°C for 12 h. Secondary antibodies used were Alexa 488 or 594-labeld antibodies (ThermoFisher, A11008 and A21203). The slides were mounted in a medium with DAPI to stain nuclei (Vector Laboratories, H1500). The stained cells were examined under a confocal microscope (Leica DM2500).

Chen S., Wang H., Li H., Zhang Y, & Wu Q. (2017). Functional Analysis of Corin Protein Domains Required for PCSK6-mediated Activation. The international journal of biochemistry & cell biology, 94, 31-39.

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Phosphorylation Analysis of STAT5a

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Cells were harvested in radioimmunoprecipitation assay buffer (RIPA) containing 1 × protease inhibitor cocktail (cOmplete, 11,697,498,001) and 1% phosphatase inhibitor cocktail II (P5726-5ML, Sigma-Aldrich, St. Louis, MO). Protein separation was achieved under reducing and denaturing conditions using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with anti-phospho-S726- or anti-phospho-S780-STAT5a antibodies obtained from Abcam (ab128896, ab30649, Cambridge, UK), anti-phospho-Y694-STAT5 antibody (9351S, Cell Signaling Technology, Danvers, MA), anti-STAT5a antibody (sc-1081X, Santa Cruz, Dallas, TX), anti-V5 tag antibody (R96025, Thermo Fisher Scientific), or anti-vinculin antibody (MCA465GA, Bio-Rad, Hercules, CA). HRP conjugated secondary antibodies were obtained from Cell Signaling Technology.

Woock A.E., Grible J.M., Olex A.L., Harrell J.C., Zot P., Idowu M, & Clevenger C.V. (2021). Serine residues 726 and 780 have nonredundant roles regulating STAT5a activity in luminal breast cancer. Scientific Reports, 11, 13506.

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Western Blot Analysis of WNT2 Proteins

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Expression of WNT2 wild-type and mutant proteins was confirmed with Western
blotting. Lysate from dual-luciferase reporter assay was additionally lysed in
buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% Triton X-100, 1 mM
EDTA, 20 mM NaF, 2 mM Na₃VO₄, and protease inhibitor
mixture (Roche, Cat#11873580001). Fifteen micrograms of extracted proteins
was separated on a 7% polyacrylamide gel and then transferred to nitrocellulose
membrane by electroblotting. Blots were simultaneously incubated with anti-V5
tag antibody at 1:2,000 (Thermo Fisher Scientific, Cat #R960-25) and
anti–β-actin antibody AC-74 at 1:2,000 (Sigma, Cat #A2228),
followed by incubation with goat anti-mouse immunoglobulin G antibody conjugated
to horseradish peroxidase (Dako Cat #P0447). Exogenous WNT2 proteins and
endogenous β-actin protein were detected by enhanced chemiluminescence
(Bio-Rad Cat #1705061) and visualized with the ChemiDoc detection system
(Bio-Rad).

Bennett M.F., Hildebrand M.S., Kayumi S., Corbett M.A., Gupta S., Ye Z., Krivanek M., Burgess R., Henry O.J., Damiano J.A., Boys A., Gécz J., Bahlo M., Scheffer I.E, & Berkovic S.F. (2022). Evidence for a Dual-Pathway, 2-Hit Genetic Model for Focal Cortical Dysplasia and Epilepsy. Neurology: Genetics, 8(1), e652.

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In Situ Proximity Ligation Assay of MuSCs

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The in situ proximity ligation assay (PLA) was performed on fixed primary proliferating MuSCs using the DuoLink PLA fluorescence technology (Sigma‐Aldrich#DUO92101), following the manufacturer′s protocol. About 2,000 isolated muscle satellite cells were seeded per well of a 96‐well plate and grown to a confluence of about 80%. Cells were fixed using 4% PFA for 7 min at room temperature. Myoblasts were then permeabilized with 0.3% Triton X‐100 in PBS three times for 5 min at room temperature. After two washing steps with PBS, cells were incubated with blocking solution in a humid chamber for 60 min at 37°C followed by incubation with primary antibodies for 1 h at room temperature. The assay was always performed with pairs of antibodies produced in mouse or rabbit, diluted 1:5,000 in antibody diluent. An anti‐V5 tag antibody (Thermo Scientific; 37–7,500) recognizing the endogenous V5‐tagged INO80 was used in combination with anti‐YY1 (Cell Signaling#46395), anti‐WDR5 (Bethyl#A302‐429A), and anti‐Ruvbl2 (Bethyl#A302‐536A) antibodies, respectively. PLA probe incubation, ligation, and signal amplification were performed according to the manufacturer's protocol. After two washing steps with PBS, DAPI was diluted 1:5,000 in PBS and added to the cells for 5 min at room temperature.

Schutt C., Hallmann A., Hachim S., Klockner I., Valussi M., Atzberger A., Graumann J., Braun T, & Boettger T. (2020). Linc‐MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. The EMBO Journal, 39(22), e105098.

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Immunofluorescence Imaging of Differentiated Neuro2a Cells

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Differentiated Neuro2a cells were fixed in 4% PFA 48 h after transfection and 24 h after differentiation. Cells were rinsed with PBS and incubated in blocking buffer (PBST supplemented with 5% NGS for 1 h, and then for 24 h with primary antibody, anti-V5 tag antibody (1:2,000, Thermo Fisher Scientific, Cat No. 46-0705), and anti–α-tubulin (YL1/2, 1:500, Santa Cruz Biotechnology, Cat No. sc53029) diluted in PBST at 4°C. Cells were washed in PBST and then incubated for 1 h with secondary antibody solution containing Alexa Fluor 488 goat anti–mouse IgG (H + L) (1:1,000; Thermo Fisher Scientific, Cat No. A11029), Alexa Fluor 568 goat anti–rat IgG (H + L) (1:1,000; Thermo Fisher Scientific, Cat No. 11007), and Hoechst 33342 (1:5,000; Thermo Fisher Scientific, Cat No. H3570) in PBST. Cells were washed in PBS and then mounted using ProLong Gold Antifade Reagent (Thermo Fisher Scientific, Cat No. P36934). The cells were imaged using a BZ-X800 microscope (Keyence). Neurite length was measured using the Neuroanatomy plugin of ImageJ software.

Kawamoto Y., Tada M., Asano T., Nakamura H., Jitsuki-Takahashi A., Makihara H., Kubota S., Hashiguchi S., Kunii M., Ohshima T., Goshima Y., Takeuchi H., Doi H., Nakamura F, & Tanaka F. (2022). Phosphorylated CRMP1, axon guidance protein, is a component of spheroids and is involved in axonal pathology in amyotrophic lateral sclerosis. Frontiers in Neurology, 13, 994676.

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Effects of Irisin and Tgfbr2 on HepG2 and AML12 Cells

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HepG2 (human hepatocellular carcinoma cell line) and AML12 (mouse hepatocyte cell line) were obtained from the American Type Culture Collection (ATCC). HepG2 and AML12 cells were cultured in Eagle’s minimum essential media and DMEM/F12 media, respectively, supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C with 5% CO₂. HepG2 cells were transfected by Fndc5/Irisin and Tgfbr2 expression plasmids using Fugene 6 reagent according to the manufacturer’s instructions. FNDC5/Irisin and Tgfbr2 protein expressions were detected by western blotting analyses using 10% SDS page gels employing anti V5 tag antibody (Thermo Fisher) and anti Tgfbr2 monoclonal antibody (Santa Cruz Biotechnology), respectively. AML12 cells in 96 well plates (seeded 5000 cells per well) were treated with irisin (1 μg/ml or 2 μg/ml) at 24 hr. Seventy-two hr after transfection (HepG2) or irisin treatment (AML12), cell viability was determined using Cell Counting Kit-8 (Dojindo).

Hori T., Yokobori K., Moore R., Negishi M, & Sueyoshi T. (2023). CAR requires Gadd45β to promote phenobarbital-induced mouse liver tumors in early stage. Frontiers in Oncology, 13, 1217847.

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Characterization of Cloned TTSuV1 Genome

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The cloned TTSuV1 genome was excised from the shuttle plasmid by Ase I digestion and circularized with DNA ligase. The porcine macrophage cell line, 3D4/31 (ATCC CRL-2844) (Weingartl et al., 2002 (link))was grown to 70% confluence in chamber slides (Lab-Tek™ II Chamber Slide™ System, Thermofisher, Grand Island, NY). Cultured cells were transfected with 400ng of the circularized genomic DNA using the Lipofectamine® LTX Reagent (Thermofisher, Grand Island, NY), following the manufacturer’s instructions. Similarly, 800ng of the ORF3 mammalian expression plasmid was transfected into the 70% confluent 3D4/31 cells. After incubation for 6, 12, 24 and 48hrs post-transfection which were the time points used for gene expression analysis, the cells were fixed in acetone: methanol (1:1) solution. The cells transfected with the genome were stained with a 1:100 dilution of a rabbit polyclonal anti-TTSuV1 antibody, (provided by Dr. X. J. Meng, Virginia Tech)(Huang et al., 2011 (link)). Cells that were transfected with the ORF3 construct were stained with a 1:500 dilution of commercial anti-V5 tag antibody (Thermofisher, Grand Island, NY), as ORF3-specific antibodies are not available. Stained slides were examined with a fluorescent microscope. The size of the ORF3 product was verified by western blotting with the anti-V5 antibody.

Singh P, & Ramamoorthy S. (2016). Lack of strong anti-viral immune gene stimulation in Torque Teno Sus Virus1 infected macrophage cells. Virology, 495, 63-70.

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