Mouse anti flag monoclonal antibody clone m2

Manufactured by Merck Group

Sourced in United States

The Mouse anti-Flag monoclonal antibody clone M2 is a laboratory reagent used to detect and purify proteins tagged with the Flag epitope. It binds specifically to the Flag peptide sequence (DYKDDDDK) and can be used in various applications such as Western blotting, immunoprecipitation, and immunoaffinity chromatography.

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Lab products found in correlation

Phalloidin tritc, by Merck Group (1 mentions) Ab186734, by Abcam (1 mentions) Sp5 confocal microscope, by Leica (1 mentions) C digit blot scanner, by LI COR (1 mentions) P0141, by Agilent Technologies (1 mentions) Westernsure chemiluminescent western blot reagent, by LI COR (1 mentions) Tl 100 instrument, by Beckman Coulter (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Alexa fluor 488 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Vectashield containing dapi, by Vector Laboratories (1 mentions) Lsm 800, by Zeiss (1 mentions) Cy3 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions) Anti ha sg77, by Thermo Fisher Scientific (1 mentions) Ha 11, by Fortrea (1 mentions)

Close spelling variants of this product

Mouse monoclonal anti-Flag (clone M2, Mouse monoclonal anti-FLAG M2 antibody Monoclonal anti‐Flag (clone M2; FLAG M2 mouse monoclonal antibody Anti-FLAG M2 monoclonal antibody Mouse anti-FLAG (clone M2, Monoclonal FLAG (clone M2, Mouse anti-FLAG M2 antibody FLAG M2 monoclonal antibody Mouse monoclonal anti-FLAG antibody

7 protocols using mouse anti flag monoclonal antibody clone m2

Quantifying Calcium-Induced Lysosome Exocytosis

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Immunofluorescence for PKD2 localization and for quantification of the number of exocytic p80 patches was performed as described previously [24] (link), [49] (link). For measurement of calcium-induced lysosome exocytosis, 10⁶ cells were allowed to attach to glass coverslips in HL5-MES medium for 3 hs, then transferred to HL5-MES containing 1 mM CaCl₂, incubated between 0 and 8 minutes as indicated, fixed with paraformaldehyde 4%, permeabilized with Triton X-100 (0.08%) and labeled with mouse monoclonal antibody anti-p80.
Mouse monoclonal antibodies against the late endosomal marker p80 (H161), the p25 marker of recycling endosomes, and the plasma membrane H36 protein, as well as a rabbit antiserum against the contractile vacuole marker Rh50 were described previously [50] (link)–[53] (link). F-actin was labeled with TRITC-phalloidin (Sigma-Aldrich). Mouse monoclonal anti-Flag antibody (clone M2) was from Sigma-Aldrich, and fluorescent secondary goat anti-mouse or anti-rabbit IgG from Molecular Probes.

Lima W.C., Vinet A., Pieters J, & Cosson P. (2014). Role of PKD2 in Rheotaxis in Dictyostelium. PLoS ONE, 9(2), e88682.

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Immunofluorescence Imaging of Transfected HeLa Cells

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After PBS washings, transfected HeLa cells grown on chamber slides were fixed with 4% PFA for 15 min. After PBS washing, cells were incubated in 50/50 acetone/methanol for 20 min. Mouse monoclonal anti-V5 antibody (R960-25, Thermo Fisher Scientific), mouse monoclonal anti-Flag antibody (clone M2, F1804, Sigma), mouse monoclonal anti-GFP antibody (clone 4B10B2, MA5-15349, Thermo Fisher Scientific), and rabbit monoclonal anti-TOM20 (ab186734, Abcam) were incubated at 1/250 for 1 h at room temperature, followed by incubation with a mouse specific Alexa-488 goat antibody (Thermo Fisher Scientific) and rabbit specific alexa-633 conjugated donkey antibody (Thermo Fisher Scientific) 1 h at room temperature in the dark. After washing, slides were mounted with Fluoromount-G imaging medium containing DAPI (Sigma). Imaging was performed using Leica SP5 confocal microscope.

Caval V., Suspène R., Khalfi P., Gaillard J., Caignard G., Vitour D., Roingeard P., Vartanian J.P, & Wain-Hobson S. (2021). Frame-shifted APOBEC3A encodes two alternative proapoptotic proteins that target the mitochondrial network. The Journal of Biological Chemistry, 297(3), 101081.

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Western Blot Analysis of DNA Repair Proteins

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Anti-XRCC4 rabbit polyclonal antibody [47 (link)], anti-FLAG mouse monoclonal antibody (clone M2; F3165; Sigma-Aldrich; St. Louis, MO, USA), anti-Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) mouse monoclonal antibody (clone 6C5; MAB374;) and anti-LIG4 guinea pig polyclonal antibody (gifted by Prof. Miki Shinohara, Kinki University) [36 (link)] were used as the primary antibody at 1/1000 to 1/5000 dilution. As the secondary antibody, horseradish peroxidase-conjugated anti-rabbit immunoglobulins swine polyclonal antibody (P0399; Dako; Glostrup, Denmark), anti-mouse immunoglobulins goat polyclonal antibody (P0447; Dako) or anti-guinea pig immunoglobulins rabbit polyclonal antibody (P0141; Dako) was used at 1/1000 to 1/3000 dilution. The immunocomplexes were developed using WesternSure Chemiluminescent Western Blot Reagent (LI-COR; Lincoln, NE, USA) and the images were captured by C-Digit Blot Scanner (LI-COR). Protein Ladder One Plus, Triple-color (Nacalai Tesque) was used as the molecular weight standard. Other procedures of western blotting followed earlier publications [31 , 32 (link)].

Asa A.D., Wanotayan R., Sharma M.K., Tsukada K., Shimada M, & Matsumoto Y. (2021). Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity. Journal of Radiation Research, 62(3), 380-389.

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Western Blotting of Viral Particles

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Western blotting was performed as described [30 (link), 36 (link)] using the following antibodies: anti-HA mouse monoclonal antibody (clone 3F10; Roche), anti-Flag mouse monoclonal antibody (clone M2; Sigma-Aldrich), anti-p24 goat antiserum (ViroStat), anti-alpha-tubulin mouse monoclonal antibody (TUBA; clone DM1A; Sigma-Aldrich); anti-hA3G rabbit antiserum (the NIH AIDS Research and Reference Reagent Program catalog number #10201); and anti-CBF-β mouse monoclonal antibody (sc-56751; Santa Cruz). For Western blotting of viral particles, 370 μl of viral supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. Transfected cells were lysed with RIPA buffer (25 mM HEPES [pH 7.4], 50 mM NaCl, 1 mM MgCl₂, 50 μM ZnCl₂, 10% glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (Roche).

Nakano Y., Yamamoto K., Ueda M.T., Soper A., Konno Y., Kimura I., Uriu K., Kumata R., Aso H., Misawa N., Nagaoka S., Shimizu S., Mitsumune K., Kosugi Y., Juarez-Fernandez G., Ito J., Nakagawa S., Ikeda T., Koyanagi Y., Harris R.S, & Sato K. (2020). A role for gorilla APOBEC3G in shaping lentivirus evolution including transmission to humans. PLoS Pathogens, 16(9), e1008812.

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Immunofluorescence Analysis of Cytoophidium

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For analysis of cytoophidium, the cells grown in 13-mm round coverslips were fixed with 4% paraformaldehyde and probed with antibodies in an indirect immunofluorescence assay, as previously described (Keppeke et al., 2018 (link); Keppeke et al., 2022 (link)).
Primary antibodies used: rabbit polyclonal anti-IMPDH2 antibody (12948-1-AP, ProteinTech), mouse anti-Myc monoclonal antibody 9E10 (sc-40, Santa Cruz Biotech), and mouse anti-Flag monoclonal antibody clone M2 (F1804, Sigma). Secondary antibodies used: Cy3-conjugated donkey anti-mouse IgG (#715-165-151, Jackson ImmunoResearch) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (#A-21206, Invitrogen). After immunofluorescence labeling, the cells were covered with VECTASHIELD containing DAPI (Vector Labs, USA), and the images were captured either by a fluorescence microscope with 200 × or 400 × magnification (Axio Imager. M2, Carl Zeiss, Germany) or by a confocal microscope (LSM 800, Carl Zeiss, Germany).

Keppeke G.D., Chang C.C., Zhang Z, & Liu J.L. (2023). Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn. Frontiers in Cell and Developmental Biology, 11, 1234592.

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Binding Assays for Leukocidin Subunits

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For binding experiments with LukS-PV, cells transiently transfected with plasmids encoding receptors with an N-terminal FLAG tag (28 (link)) were pre-labeled with a mouse anti-FLAG monoclonal antibody (clone M2, 1:500, Sigma), followed by PE-labeled goat-anti-mouse antibody (1:80, Dako). For experiments with HlgC, stably transfected cells were directly used for subsequent binding. After incubation with the protein on ice for 30 min in a volume of 50 μL, cells were washed and incubated with a FITC-conjugated mouse anti-his monoclonal antibody (1:80, LifeSpan Biosciences). Binding was detected using flow cytometry, and at least 10,000 cells were analyzed. Analysis of binding to transiently transfected cells was limited to receptor-positive cells. Binding of the S-components was expressed as the percentage of mean fluorescence in relation to saturated binding of the S-component to human C5aR1 at 31 nM.

Spaan A.N., Schiepers A., de Haas C.J., van Hooijdonk D.D., Badiou C., Contamin H., Vandenesch F., Lina G., Gerard N.P., Gerard C., van Kessel K.P., Henry T, & van Strijp J.A. (2015). Differential Interaction of the Staphylococcal Toxins Panton-Valentine Leukocidin and γ-Hemolysin CB with Human C5a Receptors. Journal of immunology (Baltimore, Md. : 1950), 195(3), 1034-1043.

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Antibody Characterization for Immunofluorescence and Western Blot

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The following antibodies were used for immunofluorescence assays (IFA) and western blot (WB) analysis at the indicated dilutions: mouse anti-HA monoclonal antibody HA.11 (Covance; 1:500 WB); rabbit polyclonal anti-HA SG77 (ThermoFisher; 1:500 IFA and WB); mouse anti-Flag monoclonal antibody clone M2 (Sigma; 1:300 IFA); mouse anti-EXP1 monoclonal antibody (Lisewski et al., 2014 (link)) (1:500 WB); mouse anti-EXP2 monoclonal antibody clone 7.7 (Hall et al., 1983 (link)) (1:500 IFA and WB); rabbit polyclonal anti-SBP1 (Blisnick et al., 2000 (link)) (1:500 IFA); mouse anti-cMYC monoclonal antibody 9E10 (ThermoFisher; 1:166 IFA and WB); rabbit polyclonal anti-Plasmodium Aldolase ab207494 (Abcam; 1:500 WB); rabbit polyclonal anti-RFP R10367 (ThermoFisher; 1:500 WB).

Nessel T., Beck J.M., Rayatpisheh S., Jami-Alahmadi Y., Wohlschlegel J.A., Goldberg D.E, & Beck J.R. (2020). EXP1 is required for organization of EXP2 in the intraerythrocytic malaria parasite vacuole. Cellular microbiology, 22(5), e13168.

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