Mouse anti v5

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Mouse anti-V5 is a monoclonal antibody that specifically recognizes the V5 epitope tag. It is commonly used in research applications to detect and purify proteins expressing the V5 tag.

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Lab products found in correlation

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166 protocols using mouse anti v5

Antibody Immunoblotting and Immunostaining Protocol

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The primary antibodies used for immunoblotting were mouse anti-V5 (1:5000, Invitrogen), guinea pig anti-Axin (1:1000, [17 (link)]), rabbit anti-Kinesin Heavy Chain (1:10000, Cytoskeleton), mouse anti-alpha-Tubulin (1:10000, DM1A, Sigma), rabbit anti-alpha-Tubulin (1:10000, Sigma), rabbit anti-Gluthathione-S-Transferase (1:10000, Invitrogen), rabbit anti-phospho-LRP6 [Thr1572] (1:1000, Millipore), mouse anti-Nervana antibody (Nrv5F7, 1:1000, DSHB), and guinea pig anti-Arrow antibody (1:1000, [74 (link)]). The primary antibodies used for immunostaining were guinea pig anti-Axin (1:1000, [17 (link)]), rabbit anti-β-gal (1:1000; MP Biomedicals), mouse anti-Arm (1:20; DSHB), mouse anti-Fas III (1:20; DSHB), mouse anti-Discs Large (1:20; DSHB), rabbit anti-GFP (1:200; Invitrogen), mouse anti-V5 (1:5000; Invitrogen), rabbit anti-Apc2 (1:1000; [75 (link)]), and guinea pig anti-Senseless (1:1000, [76 (link)]).
The secondary antibodies used for immunoblotting were: goat anti-rabbit HRP conjugate (1:10000, Biorad), goat anti-mouse HRP conjugate (1:10000, Biorad), and goat anti-guinea pig HRP conjugate (1:10000, Jackson ImmunoResearch). The secondary antibodies used for immunostaining were goat or donkey Alexa Fluor 488, 555 or Cy5 conjugates (1:400; Invitrogen).

Wang Z., Tacchelly-Benites O., Yang E, & Ahmed Y. (2016). Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling. PLoS Genetics, 12(12), e1006494.

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Antibody Immunostaining and Immunoblotting Protocol

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The primary antibodies used for immunostaining were mouse anti-V5 (1:5000; Invitrogen), mouse anti-Wingless (1:200, 4D4 concentrated antibody, Developmental Studies Hybridoma Bank, DSHB). The secondary antibodies used for immunostaining were goat or donkey Alexa Fluor 488 or 555 conjugates (1:400; Invitrogen). The primary antibodies used for immunoblotting were mouse anti-V5 (1:5000, Invitrogen), guinea pig anti-Axin (1:1000, [54 (link)]), rabbit anti-Kinesin Heavy Chain (1:10000, Cytoskeleton), mouse anti-Arm (1:100, N2 7A1, DSHB), mouse anti-alpha-Tubulin (1:10000, DM1A, Sigma), rabbit anti-alpha-Tubulin (1:10000, Sigma), rabbit anti-Gluthathione-S-Transferase (1:10000, Invitrogen), guinea pig anti-Apc2 (GP10) 1:5000 [44 (link)]; rabbit anti-phospho-LRP6 [Thr1572] (1:1000, Millipore), rat anti-Dishevelled (1:1000, [70 (link)]), and guinea pig anti-Arrow (1:1000, [71 (link)]). The secondary antibodies used for immunoblotting were: goat anti-rabbit HRP conjugate (1:10000, Biorad), goat anti-mouse HRP conjugate (1:10000, Biorad), and goat anti-guinea pig HRP conjugate (1:10000, Jackson ImmunoResearch).

Tacchelly-Benites O., Wang Z., Yang E., Benchabane H., Tian A., Randall M.P, & Ahmed Y. (2018). Axin phosphorylation in both Wnt-off and Wnt-on states requires the tumor suppressor APC. PLoS Genetics, 14(2), e1007178.

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Phosphorylation Detection via Phos-tag SDS-PAGE

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Phos-tag SDS-PAGE and Western blotting were performed according to previous studies (Liu et al. 2017 (link)). In brief, Phos-tag SDS-PAGE gels were made adding 30 μM Phos-tag reagent according to the manufacturer’s protocol (Wako Chemical). Samples were resolved on SDS-PAGE with Phos-tag and detected via Western blotting mouse anti-V5 (Life Technologies, R960-25) and IRDye 680 goat anti-mouse (LI-COR, 926-32220). Bands were quantified using Image studio lite software (Li-COR). All bands that migrate slower than the most intensive band were considered as putative phosphorylated forms. A small subset of these bands displayed insulin/rapamycin responsiveness.

Liu Y., Mattila J, & Hietakangas V. (2020). Systematic Screen for Drosophila Transcriptional Regulators Phosphorylated in Response to Insulin/mTOR Pathway. G3: Genes|Genomes|Genetics, 10(8), 2843-2849.

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Proximity Ligation Assay in HEK293T Cells

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HEK293T (ATCC) cells, whose morphology was used for identification and are routinely used in the lab, were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) and supplemented with L-glutamine, penicillin/streptomycin (ThermoFisher Scientific), and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA), and, one day before transfection, plated on glass coverslips in a 24-well plate at a density of 0.15 million cells/mL. Constructs were transfected using standard calcium-phosphate protocol. Transgenes were under the control of a UAS promoter and co-transfected with a CMV-Gal4 construct. Cells were fixed with 4% paraformaldehyde in 1X PBS approximately 40 hr post transfection. PLA was performed using Duolink/PLA reagents (Sigma-Aldrich), according to the manufacturer’s protocol. The blocking solution used was 5% normal goat serum in 0.3% Triton-X solution in 1X PBS. The following primary antibodies were used: mouse anti-V5 (Life Technologies (RRID:AB_2556564); at 1:500); rabbit anti-GFP (Life Technologies (RRID:AB_221569); at 1:1000). Cells were imaged using the Zeiss LSM700 confocal microscope with a 63x oil objective and images were acquired using the Zen software; GFP+ cells were counted manually and scored as PLA+ if they had more than three puncta of PLA reaction product.

Lee G, & Schwarz T.L. (2016). Filamin, a synaptic organizer in Drosophila, determines glutamate receptor composition and membrane growth. eLife, 5, e19991.

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Immunofluorescence Microscopy of Chromosome Spreads

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Chromosome spreads were performed as previously described (Wahba et al., 2013 (link)). Slides were incubated with a mouse anti-FLAG monoclonal (Sigma-Aldrich) at 1:2000, mouse anti-V5 (Life Technologies, Carlsbad, CA) at 1:2000, polyclonal rabbit anti-Pds5 at 1:2000, or polyclonal rabbit anti-Mcd1 at 1:2000 dilutions. The primary antibody was diluted in blocking buffer (5% bovine serum albumin, 0.2% milk, 1× phosphate-buffered saline, and 0.2% Triton X-100). The secondary Cy3-conjugated goat anti-mouse antibody (115-165-003) was obtained from Jackson ImmunoResearch (West Grove, PA) and diluted 1:2000 in blocking buffer. Indirect immunofluorescence was observed using an Olympus IX-70 microscope with a 100×/NA 1.4 objective and Orca II camera (Hamamatsu, Bridgewater, NJ).

Eng T., Guacci V, & Koshland D. (2014). ROCC, a conserved region in cohesin's Mcd1 subunit, is essential for the proper regulation of the maintenance of cohesion and establishment of condensation. Molecular Biology of the Cell, 25(16), 2351-2364.

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Antibody Panel for N-terminal Acetyltransferases

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The following antibodies were used: rabbit anti-Naa10 (Abcam #ab155687), rabbit anti-Naa10 (Protein Tech #14803-1-AP), rabbit monoclonal anti-NAA10 (Cell Signaling, #13357), goat anti-Naa10 (Santa Cruz, #sc-33256), rabbit anti-Naa10 (Santa Cruz, #sc-33820), rabbit anti-Naa11 (Novus Biologicals; #NBP1-90853), mouse anti-Naa15/NARG1 (Abcam; #ab60065), rabbit polyclonal anti-NAA15 (Arnesen et al., 2005 (link)), rabbit anti-Naa50 (LifeSpan BioSciences; #LS-C81324-100), rabbit anti-FLAG (Sigma; #F7425), mouse anti-GAPDH (Abcam; #ab9484), goat anti-actin (Santa Cruz, #1615), mouse anti-GST (GenScript; #A00865), and mouse anti-V5 (Life Technologies; #R960-25). The antibody against the potential mNaa10 paralog mNaa12 (Gm16286, UniProt: Q9CQX6) was raised in rabbits after immunization with a synthetic peptide of the Naa12 C-terminus (aa191-205: QENLAGGDSGSDGKD-C) conjugated to OVA by PrimmBiotech.

Kweon H.Y., Lee M.N., Dorfel M., Seo S., Gottlieb L., PaPazyan T., McTiernan N., Ree R., Bolton D., Garcia A., Flory M., Crain J., Sebold A., Lyons S., Ismail A., Marchi E., Sonn S.K., Jeong S.J., Jeon S., Ju S., Conway S.J., Kim T., Kim H.S., Lee C., Roh T.Y., Arnesen T., Marmorstein R., Oh G.T, & Lyon G.J. (2021). Naa12 compensates for Naa10 in mice in the amino-terminal acetylation pathway. eLife, 10, e65952.

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Co-immunoprecipitation of β-actin and V5-PFN1

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Co-immunoprecipitation of ß-actin with V5-PFN1 in lysates of HEK293 cells was performed as previously described [2 (link)]. For Western blot detection of V5-PFN1 and ß-actin we used mouse anti-V5 (Life Technologies Cat# R96025 RRID:AB_10050879) and rabbit anti-ß-actin (Cell Signaling Technology Cat# 4970 RRID:AB_2223172) antibodies. Secondary antibodies were goat anti-mouse IgG-HRP (Life Technologies Cat# G21040 RRID:AB_11180340) and goat anti-rabbit IgG-HRP (Life Technologies Cat# G21234 RRID:AB_10837906), respectively.

Freischmidt A., Schöpflin M., Feiler M.S., Fleck A.K., Ludolph A.C, & Weishaupt J.H. (2015). Profilin 1 with the amyotrophic lateral sclerosis associated mutation T109M displays unaltered actin binding and does not affect the actin cytoskeleton. BMC Neuroscience, 16, 77.

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Western Blotting Protein Detection

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Western blot analysis was performed essentially as previously described (Vucetic et al., 2008 (link); Zhao et al., 2009 (link)). Primary antibodies used were goat anti-GAPDH (sc-48176, Santa Cruz), mouse anti-V5 (R960-25, Life Technologies), mouse anti-FLAG (F1804, Sigma-Aldrich,), mouse anti-H3 histone (10799, Abcam) and rabbit anti-HP1α (2616, Cell Signaling, Beverly, MA). Secondary antibodies used were donkey anti-mouse IRDye 800CW, donkey anti-rabbit IRDYE 800CW, and donkey anti-goat IRDye 680CW purchased from LI-COR, Lincoln, NE. Images were captured and quantitated using the LI-COR Odyssey instrument and software. GAPDH levels were used as the loading control.

Wang F., Wendling K.S., Soprano K.J, & Soprano D.R. (2014). The SAP Motif and the C-terminal RS- and RD/E-rich Region Influences the Sub-Nuclear Localization of Acinus Isoforms. Journal of cellular biochemistry, 115(12), 2165-2174.

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Microscopic Imaging of S2 Cells

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S2 cells were fixed and processed as previously described (Rogers and Rogers, 2008 (link)) by spreading S2 cells on concanavalin A–coated glass-bottom dishes and fixing with ice-cold methanol. Primary antibodies were diluted to concentrations ranging from 1 to 20 µg/ml. They included rabbit anti–PLP (Rogers et al., 2009 (link)), guinea pig anti-Asl (Klebba et al., 2013 (link)), rat anti-Cep135, rat anti–Asl NT, and mouse anti-V5 (Life Technologies) antibodies. Goat secondary antibodies (conjugated with Cy2, Rhodamine red-X, or Cy5 [Jackson ImmunoResearch]) were used at 1:1,500. Hoechst 33342 (Life Technologies) was used at a final concentration of 3.2 µM. Cells were mounted in 0.1 M n-propyl galate, 90% (by volume) glycerol, and 10% PBS solution. Specimens were imaged using a DeltaVision Core deconvolution system (Applied Precision) equipped with an Olympus IX71 microscope, a 100× objective (NA 1.4), and a cooled CCD camera (CoolSNAP HQ2; Photometrics). Images were acquired with softWoRx v.1.2 software (Applied Science). Superresolution microscopy was performed using a Zeiss ELYRA S1 (SR-SIM) microscope equipped with an AXIO Observer Z1 inverted microscope stand with transmitted (HAL), UV (HBO), and solid-state (405/488/561-nm) laser illumination sources, a 100× objective (NA 1.4), and EM-CCD camera (Andor iXon). Images were acquired with ZEN 2011 software.

McLamarrah T.A., Buster D.W., Galletta B.J., Boese C.J., Ryniawec J.M., Hollingsworth N.A., Byrnes A.E., Brownlee C.W., Slep K.C., Rusan N.M, & Rogers G.C. (2018). An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly. The Journal of Cell Biology, 217(4), 1217-1231.

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Immunohistochemistry Profiling of Neural Markers

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Immunohistochemistry was performed as previously described (Lammel et al., 2012). Briefly, animals were intracardially perfused with 4% paraformaldehyde in PBS and post-fixed at 4 °C overnight. 50 µm coronal sections were sliced on a Leica VT1000 vibratome. Primary antibodies used were: guinea pig anti-Vglut1 (1:250; Millipore), guinea pig anti-Vglut2 (1:1000; Millipore), rabbit anti-TH (1:1000; Millipore), rabbit anti-NeuN (1:2000; Abcam), mouse anti-GAD65 (1:250; DSHB U. of Iowa), guinea pig anti-substance P (1:500; Abcam). For spaghetti monster experiments, epitope antibodies used were: rabbit anti-FLAG (1:1000; Sigma), goat anti-myc (1:1000; Abcam), mouse anti-V5 (1:1000; Life Technologies), rat anti-HA (1:1000; Roche). Alexa Fluor dyes conjugated to either 405, 488, 568, or 647 were used for secondary antibodies (Life Technologies, Abcam). All dilutions used were 1:1000 except for Alexa Fluor 405 at 1:500. All images were acquired with an Olympus FluoView FV1200 confocal microscope.

Knowland D., Lilascharoen V., Pacia C.P., Shin S., Wang E.H, & Lim B.K. (2017). Distinct ventral pallidal neural populations mediate separate symptoms of depression. Cell, 170(2), 284-297.e18.

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