Mouse anti v5 tag

Manufactured by Thermo Fisher Scientific

Sourced in Japan

The Mouse anti-V5 tag is a primary antibody that specifically recognizes the V5 epitope tag. The V5 tag is a short peptide sequence (GKPIPNPLLGLDST) that can be added to recombinant proteins to facilitate detection and purification. The Mouse anti-V5 tag antibody can be used to detect and immunoprecipitate V5-tagged proteins.

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Lab products found in correlation

Mouse anti β actin, by Merck Group (2 mentions) Mouse anti flag m2, by Merck Group (2 mentions) Rabbit anti eif4e2 4ehp, by Proteintech (2 mentions) Rabbit anti ddx6, by Proteintech (2 mentions) Rabbit anti cnot9, by Proteintech (2 mentions) Rabbit anti gigyf2, by Proteintech (2 mentions) Rabbit anti znf598, by Thermo Fisher Scientific (2 mentions) Mouse anti 6xhis tag, by Thermo Fisher Scientific (2 mentions) Mouse anti gapdh, by Proteintech (2 mentions) Anti mouse, by GE Healthcare (1 mentions) Alexa fluor 546 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Rat anti ha clone 3f10, by Merck Group (1 mentions) Mouse anti flag clone m2, by Merck Group (1 mentions) Rabbit anti pin1, by Cell Signaling Technology (1 mentions) Tgx gradient gel, by Bio-Rad (1 mentions) Anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Image quant las4000 mini apparatus, by GE Healthcare (1 mentions) Chemi lumi one super, by Nacalai Tesque (1 mentions) Rabbit anti β actin monoclonal antibody, by Cell Signaling Technology (1 mentions) Mouse monoclonal anti flag m2, by Merck Group (1 mentions)

6 protocols using mouse anti v5 tag

Antibodies for Western Blot and IP

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Primary antibodies used in Western blot and IP experiments were rat anti-HA clone 3F10 (Sigma-Aldrich), mouse anti-Flag clone M2 (Sigma-Aldrich), mouse anti-V5 Tag (Thermo Fisher Scientific), mouse anti-TRIM8 (Santa Cruz Biotechnology, sc-398878), mouse anti-IRF7 clone G-8 (Santa Cruz Biotechnology, sc-74472), rabbit anti-pIRF7 (Ser⁴⁷⁷) clone D7E1W (no. 12390, Cell Signaling), rabbit anti-ubiquitin (linkage-specific K63) clone EPR8590-448 (Abcam), rabbit anti-Pin1 (no. 3722, Cell Signaling), and mouse anti–β-actin (Sigma-Aldrich). Secondary antibodies used were anti-mouse, anti-rat, and anti-rabbit horseradish peroxidase (HRP)–conjugated (GE Healthcare Life Sciences).
For immunofluorescence and flow cytometry, we used Alexa Fluor 488 mouse anti-IRF7 (pS477/pS479) clone K47-671 (BD Biosciences), mouse anti–IFN-α phycoerythrin (PE) clone LT27:295 (Miltenyi Biotec), mouse allophycocyanin (APC) anti–BDCA-4 clone REA380 (Miltenyi Biotec), mouse fluorescein isothiocyanate (FITC) anti-CD123 clone AC145 (Miltenyi Biotec), and rabbit anti-TRIM8 (Thermo Fisher Scientific) antibodies. Secondary antibody (for TRIM8 labeling) was Alexa Fluor 546 goat anti-rabbit (Thermo Fisher Scientific).

Maarifi G., Smith N., Maillet S., Moncorgé O., Chamontin C., Edouard J., Sohm F., Blanchet F.P., Herbeuval J.P., Lutfalla G., Levraud J.P., Arhel N.J, & Nisole S. (2019). TRIM8 is required for virus-induced IFN response in human plasmacytoid dendritic cells. Science Advances, 5(11), eaax3511.

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Immunoblotting Proteins with Diverse Antibodies

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Proteins were separated by SDS-PAGE on 4–15% TGX gradient gels (Bio-Rad) and transferred onto PVDF membranes. The membranes were blocked in PBS containing 5% non-fat milk and 0.1% Tween 20 for 30 min at room temperature. Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-V5 tag (Thermo Fisher Scientific, R96025), rabbit anti-ZNF598 (Thermo Fischer Scientific, 703,601) and mouse anti-6xHis Tag (Thermo Fischer Scientific, MA1-21315-HRP).

Zou L., Moch C., Graille M, & Chapat C. (2022). The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex. iScience, 25(7), 104646.

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Protein Expression Analysis in HEK293FT Cells

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HEK293FT cells were seeded in 24-well cluster plates and transfected with 1 μg pLenti6-E/3a/M-V5His, pLenti-GFP (green fluorescent protein), or pLenti-M2 using polyethylenimine (PEI) Max. At 24 h post-transfection, the cells were lysed with RIPA buffer (50 mM Tris–HCl, 1% NP-40, 0.05% sodium dodecyl sulfate (SDS), 150 mM NaCl and 1 mM EDTA). And the lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) followed by electroblotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated over night with mouse anti-V5-tag (R960-25, Invitrogen), mouse anti-influenza A virus M2 (14C2, Abcam), mouse anti-GFP (GF200, Nacalai Tesque), or rabbit anti-tubulin (DM1A, Santa Cruz) antibodies, followed by horseradish peroxide-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories) or anti-rabbit IgG (Invitrogen). After washing 3 times with washing buffer (0.05% Tween-20/PBS), the membranes were exposed using Chemi-Lumi One Super (Nacalai Tesque), and the chemiluminescent signals were captured by an ImageQuant LAS-4000 mini apparatus (GE Healthcare).

Chen I.Y., Moriyama M., Chang M.F, & Ichinohe T. (2019). Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome. Frontiers in Microbiology, 10, 50.

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SARS-CoV-2 N Antibody Detection

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The antibodies used in this study include: Immunofluorescence: rabbit-anti-SARS-CoV-2 N antibody (gift from Kwok-Yung Yuen, University of Hong Kong), mouse anti-HM1.24 (BST2) (a gift from Chugai Pharmaceutical Co., Kanagawa, Japan), rat anti-FLAG-AlexaFluor-488 (Biolegend, #637317), mouse anti-HA-AlexaFluor-594 (Biolegend, #901511), donkey anti-mouse-AlexaFluor-488 (Jackson ImmunoResearch, #715-545-150), donkey anti-mouse-Rhodamine-Red-X (Jackson ImmunoResearch, #715-295-150), and mouse anti-V5 tag (Invitrogen, R960-25). Western blotting: mouse monoclonal anti-Orf7a (GeneTex #GTX632602), rabbit polyclonal anti-BST2 (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: Anti-BST-2 Polyclonal (cat #11721) from Drs. Klaus Strebel and Amy Andrew), mouse monoclonal anti-V5 tag (Invitrogen, #R960-25), mouse monoclonal anti-GAPDH (GeneTex, #GTX627408), mouse monoclonal anti-FLAG M2 (Sigma, #F1804), rabbit monoclonal anti-β-actin antibody (Cell Signaling, #4970) and rabbit monoclonal anti-CoxIV antibody (Cell Signaling #4850).

Martin-Sancho L., Lewinski M.K., Pache L., Stoneham C.A., Yin X., Becker M.E., Pratt D., Churas C., Rosenthal S.B., Liu S., Weston S., De Jesus P.D., O’Neill A.M., Gounder A.P., Nguyen C., Pu Y., Curry H.M., Oom A.L., Miorin L., Rodriguez-Frandsen A., Zheng F., Wu C., Xiong Y., Urbanowski M., Shaw M.L., Chang M.W., Benner C., Hope T.J., Frieman M.B., García-Sastre A., Ideker T., Hultquist J.F., Guatelli J, & Chanda S.K. (2021). Functional landscape of SARS-CoV-2 cellular restriction. Molecular Cell, 81(12), 2656-2668.e8.

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Antibody and Knockdown Assay Protocol

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The following antibodies were used: rabbit anti-eIF4E2 (4EHP) (Genetex, GTX103977), mouse anti-eIF4E (BD Biosciences, 610270), rabbit anti-eIF4ENIF1 (4E-T; abcam, ab55881), rabbit anti-DDX6 (Bethyl Laboratories, A300-460A), rabbit anti-CNOT1 (Proteintech, 14276–1-AP), mouse anti-α-Tubulin (Santa Cruz, sc-23948), mouse anti-β-actin (Sigma, A5441), mouse anti-Flag (Sigma, F3165), rabbit anti-HA (Sigma, H6908), mouse anti-V5 tag (Invitrogen, R960-25), rabbit anti-PARP (Cell Signaling Cat# 9532S), rabbit anti-DUSP6 (abcam Cat# ab76310), rabbit anti-DUSP7 (abcam Cat# ab100921),), rabbit anti-CNOT9 (RQCD1) (Proteintech Cat# 22503–1-AP), mouse GAPDH (Santa Cruz, sc-32233), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Cat#4370), mouse anti-MEK1/2 (Cell Signaling Cat# 4694S), rabbit anti-phospho-MEK1/2 (Ser217/221; Cell Signaling Cat# 9121S), rabbit anti-phospho-RPS6 (Ser240/244) (Cell Signaling Cat# 2215), and mouse anti-RPS6 (C-8).
The following siRNA and shRNAs were used: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05), 4EHP siRNA SMARTpool (Dharmacon, L-019870–01), eIF4ENIF1 (4E-T) siRNA SMARTpool (Dharmacon, L-013237–01), CNOT1 siRNA SMARTpool (Dharmacon, L-015369–01-0005), CNOT9 siRNA SMARTpool (Dharmacon, L-019972–00), Non-Targeting shRNA Controls (Sigma, SHC002), and EIF4E2 shRNA (Sigma, TRCN0000152006).

Jafarnejad S.M., Chapat C., Matta-Camacho E., Gelbart I.A., Hesketh G.G., Arguello M., Garzia A., Kim S.H., Attig J., Shapiro M., Morita M., Khoutorsky A., Alain T., Gkogkas C.G., Stern-Ginossar N., Tuschl T., Gingras A.C., Duchaine T.F, & Sonenberg N. (2018). Translational control of ERK signaling through miRNA/4EHP-directed silencing. eLife, 7, e35034.

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Immunoblotting of Protein Complexes

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Proteins were separated by SDS-PAGE on 4 -15 % gradient gels (BioRad) and transferred onto PVDF membranes. The membranes were blocked in PBS containing 5 % non-fat milk and 0.1 % Tween 20 for 30 min at room temperature. Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP), mouse anti-GAPDH (Proteintech, 60004-1-Ig), mouse anti-V5 tag (Invitrogen, R96025), rabbit anti-ZNF598 (Thermo Fischer Scientific, 703601) and mouse anti-6xHis Tag (Thermo Fischer Scientific, MA1-21315-HRP).

Zou L., Moch C., Graille M., & Chapat C. (2022). The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells.

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