Mouse monoclonal anti flag clone m2

Manufactured by Merck Group

Sourced in Germany, United Kingdom, Spain

The Mouse monoclonal anti-Flag (clone M2) is a laboratory reagent used for the detection and purification of proteins tagged with the Flag epitope. It is a mouse-derived monoclonal antibody that specifically binds to the Flag peptide sequence (DYKDDDDK).

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11 protocols using mouse monoclonal anti flag clone m2

Western Blot Antibody Reagents

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Mouse monoclonal anti-flag (M2 clone; #F1804) and anti-ß-actin (clone AC-15; #A1978) were purchased from Sigma (Castle Hill, NSW). Rabbit polyclonal anti-Mcl-1 (#600-401-394) was purchased from Rockland (Jomar Bioscience, Welland, SA). Hamster monoclonal anti-Bcl-2 (clone 3F11) was obtained from Andreas Strasser (WEHI). Anti-Bcl-w and anti-Bcl-xL were obtained from Lorraine O'Reilly (WEHI) and rat anti-A1 (clone 6D6) was provided by David Huang (WEHI). Cycloheximide (#C7698), 4-hydroxytamoxifen (4HT; #H7904), roscovitine (#R7772) and MG132 (#C2211) were purchased from Sigma.

Goodall K.J., Finch-Edmondson M.L., van Vuuren J., Yeoh G.C., Gentle I.E., Vince J.E., Ekert P.G., Vaux D.L, & Callus B.A. (2016). Cycloheximide Can Induce Bax/Bak Dependent Myeloid Cell Death Independently of Multiple BH3-Only Proteins. PLoS ONE, 11(11), e0164003.

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Subcellular Localization of ATP7A

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Immunostaining was performed as previously described9 (link). Cells were cultured on collagen-coated cover slips in a 12-well plate, fixed with 3.7% paraformaldehyde in PBS for 20 min, permealized with 0.2% (w/v) Triton X-100 in PBS for 10 min, and blocked with 1% (w/v) BSA/1% (w/v) gelatin in PBS. Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290). Secondary antibodies used for cell staining were: goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001), goat polyclonal anti-rabbit IgG Alexa555-conjugate (Molecular Probes, A-21428). Cells were imaged with a Zeiss LSM 710. Images were processed with ZEN and Image J. Anti-EEA1, anti-Rab11, and anti-Lamp1 antibodies were kindly provided by Dr Rajini Rao (Johns Hopkins University). All antibodies were used at a dilution of 1:200.

Hatori Y., Yan Y., Schmidt K., Furukawa E., Hasan N.M., Yang N., Liu C.N., Sockanathan S, & Lutsenko S. (2016). Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway. Nature Communications, 7, 10640.

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Antibody Western Blot Protocol

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The following antibodies were used as primary antibodies: mouse monoclonal anti-FLAG (clone M2; Sigma), mouse monoclonal anti-6×His (Clontech), rabbit polyclonal anti-IgaA (42 (link)), rabbit polyclonal anti-OmpA (gift from H. Schwarz, University of Tübingen, Germany), and rabbit polyclonal anti-PBP3 (our lab collection). Goat polyclonal anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase was used as a secondary antibody (Bio-Rad). SDS-PAGE and Western blotting were performed as described previously (33 (link)).

Castanheira S., Cestero J.J., Rico-Pérez G., García P., Cava F., Ayala J.A., Pucciarelli M.G, & García-del Portillo F. (2017). A Specialized Peptidoglycan Synthase Promotes Salmonella Cell Division inside Host Cells. mBio, 8(6), e01685-17.

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Generation and Characterization of CHMP4C Phospho-Specific Antibodies

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CHMP4C phospho-specific antibodies were raised in rabbits against synthetic CHMP4C peptides encompassing residues 206–220 (TARRSRAASSQRAEEC) and containing either a phosphorylated serine at position S210 (TARRpSRAASSQRAEEC; mono-phospho CHMP4C) or phosphorylated serines at position S210, S214 and S215 (TARRpSRAApSpSQRAEEC; triphospho CHMP4C). Sera were first eluted through a column containing a non-phosphorylated peptide, and then each antibody was affinity purified using the appropriate phosphopeptide. Peptide synthesis, conjugation, rabbit immunizations, serum production and affinity purification were carried out by Generon, UK.
Other antibodies used in this study were: mouse monoclonal anti α-tubulin (clone DM1A, Sigma, T9026), chicken polyclonal anti-α-tubulin (Abcam, ab89984), mouse monoclonal anti-Flag (clone M2, Sigma, F3165), rabbit polyclonal anti-Aurora B (Abcam, ab2254), mouse monoclonal anti-Borealin (clone 1D11-MLB Life Science M147-3), rabbit polyclonal anti-KIF23/MKLP1 (clone N19, Santa Cruz Biotechnology, sc-867), rabbit polyclonal anti-CHMP4B, a gift of Sagona & Stenmark [36 (link)]. Peroxidase- and Alexa-fluor-conjugated secondary antibodies were purchased from Jackson Laboratories and Thermo, respectively.

Capalbo L., Mela I., Abad M.A., Jeyaprakash A.A., Edwardson J.M, & D'Avino P.P. (2016). Coordinated regulation of the ESCRT-III component CHMP4C by the chromosomal passenger complex and centralspindlin during cytokinesis. Open Biology, 6(10), 160248.

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Diverse Antibody Utilization in Research

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We utilized the following antibodies: mouse monoclonal anti-FLAG (clone M2, Sigma-Aldrich), rabbit polyclonal anti-FLAG (Sigma-Aldrich), rabbit polyclonal anti-Senataxin (Novus Biologicals, NBP1-94712), rabbit polyclonal anti-Senataxin (kind gift from Domenico Delia, University of Milan, Milan, Italy), mouse monoclonal anti-HA (BioLegend), rabbit monoclonal anti-p63-α (clone D2K8X; Cell Signaling Technology), mouse monoclonal anti-DNA–RNA hybrids clone S9.6 (Millipore), mouse monoclonal anti-vinculin (Sigma), rabbit polyclonal anti‐KRT1 (BioLegend, PRB‐149P), rabbit polyclonal anti‐KRT10 (Covance PRB159P), rabbit polyclonal anti ZNF750 (Sigma-Aldrich, HPA023012), rabbit polyclonal anti-Loricrin (Covance PRB145P), and rabbit polyclonal anti-Adenosine (Biovision).

Gatti V., Fierro C., Compagnone M., La Banca V., Mauriello A., Montanaro M., Scalera S., De Nicola F., Candi E., Ricci F., Fania L., Melino G, & Peschiaroli A. (2022). ΔNp63-Senataxin circuit controls keratinocyte differentiation by promoting the transcriptional termination of epidermal genes. Proceedings of the National Academy of Sciences of the United States of America, 119(10), e2104718119.

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Antibody Sources for Immunoblotting

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Antibodies used for immunoblotting were as follows: mouse monoclonal anti-hamster HMGCR IgG-A9 (CRL-1811, ATCC, 2 μg ml⁻¹), mouse monoclonal anti-hamster SREBP-2 IgG-7D4 (CRL-2198, ATCC, 1 μg ml⁻¹), mouse monoclonal anti-clathrin heavy chain (610500, BD Biosciences, 1:1000), mouse monoclonal anti-ubiquitin antibody P4D1 (SC-8017, Santa Cruz Biotechnology, 1:1000), mouse monoclonal anti-HA (clone HA-7) (H3663, Sigma-Aldrich, 1:1000), mouse monoclonal anti-Flag (clone M2) (F3165, Sigma-Aldrich, 1:1000), mouse monoclonal anti-T7 (69522, Novagen, 1:5000). Rabbit polyclonal antibody H2 against HMGCR (1 μg ml⁻¹) and GFP (0.2 μg ml⁻¹) were prepared in our laboratory^{65 (link)}. Horseradish peroxidase-conjugated goat anti-mouse (115-035-003, 1:5000) and anti-rabbit (111-035-144, 1:5000) secondary antibodies were from Jackson ImmunoResearch Laboratories.

Jiang S.Y., Li H., Tang J.J., Wang J., Luo J., Liu B., Wang J.K., Shi X.J., Cui H.W., Tang J., Yang F., Qi W., Qiu W.W, & Song B.L. (2018). Discovery of a potent HMG-CoA reductase degrader that eliminates statin-induced reductase accumulation and lowers cholesterol. Nature Communications, 9, 5138.

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Immunostaining of Murine Testicular Tissue

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Testes from 2 to 4-day-old males were dissected in PBS-T (PBS 1X with 0.15% Triton X-100) and fixed for 20 min in 4% PFA at room temperature. Testes were washed 3 times in PBS-T and incubated with primary antibody overnight at 4°C. After three 20 minutes washes in PBS-T, they were incubated with secondary antibodies at room temperature for 2 hours. Testes were then mounted in Dako mounting medium (Agilent, #S3023) containing 1 μg/ml DAPI.
Primary antibodies used were mouse monoclonal anti-V5 (Invitrogen #R960-25, 1:500), mouse monoclonal anti-Flag (clone M2—Sigma-Aldrich #F3165, 1:1000) and rabbit polyclonal anti-acetyl Histone H4 (Merck Millipore #06–589; 1:500). Secondary antibodies were used at a 1:1000 dilution and included: Alexa Fluor 647 goat anti-rabbit IgG (Invitrogen, # AB-2535813), Alexa Fluor 555 goat anti-mouse IgG1 (Invitrogen #AB-2535769) and Alexa Fluor 488 goat anti-mouse IgG2a (Invitrogen #AB-2535771). Images were acquired on an LSM 800 confocal microscope equipped with a 40X 1.4 NA objective lens (Carl Zeiss). Images were processed with Image J and GIMP.

Terretaz K., Horard B., Weill M., Loppin B, & Landmann F. (2023). Functional analysis of Wolbachia Cid effectors unravels cooperative interactions to target host chromatin during replication. PLOS Pathogens, 19(3), e1011211.

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SDS-PAGE and Western Blot Analysis

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To prepare cell extracts, cells were lysed in 1× Laemmli buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, 100 mM dithiothreitol, 0.02% bromophenol blue). Viruses, VLPs, and lung homogenates were mixed with 5× Laemmli buffer to obtain 1× Laemmli buffer lysates. Samples were run on SDS-PAGE gels and blotted onto nitrocellulose membranes (Hybond ECL; GE Healthcare). All stainings were performed in Tris-buffered saline mixed with 0.5% Tween-20 (Sigma-Aldrich) and 3% powdered milk. The following antibodies were used: mouse monoclonal anti-Flag (clone M2; Sigma-Aldrich), rabbit polyclonal anti-IFITM3 (Proteintech), mouse monoclonal anti–β-actin (Santa Cruz Biotechnology), mouse monoclonal anti–HIV-1 p24 (ab9071; Abcam), mouse monoclonal anti-IAV M1 (HB-64; American Type Culture Collection), rabbit polyclonal anti-nucleoprotein (NP; a kind gift of A. Nieto, Spanish National Center for Biotechnology, Madrid, Spain), mouse monoclonal anti-A/WSN/33 HA (clone H15-B9-22 from Wistar), rabbit polyclonal anti-A/WSN/33, and mouse monoclonal anti-A/Netherlands/602/2009 HA clone 31C2 (Manicassamy et al., 2010 (link)). Secondary antibody staining was performed using near-infrared fluorescent secondary antibodies (Li-Cor) and images were acquired on an Odissey Fc imaging system. Western blot signal intensities were quantified using the Image Studio software (Li-Cor).

Lanz C., Schotsaert M., Magnus C., Karakus U., Hunziker A., Sempere Borau M., Martínez-Romero C., Spieler E.E., Günther S.C., Moritz E., Hale B.G., Trkola A., García-Sastre A, & Stertz S. (2021). IFITM3 incorporation sensitizes influenza A virus to antibody-mediated neutralization. The Journal of Experimental Medicine, 218(6), e20200303.

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Antibody Panel for Cell Signaling

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The following antibodies were used in this study: mouse monoclonal anti α-tubulin (clone DM1A, Sigma, T9026), chicken polyclonal anti-α-tubulin (Abcam, ab89984), mouse monoclonal anti-Flag (clone M2, Sigma, F3165), mouse monoclonal anti-CIT-K (BD Transduction Laboratories, 611377), rabbit polyclonal anti-Aurora B (Abcam, ab2254), rabbit polyclonal anti-pT232 Aurora B (Abcam, ab61074), rabbit polyclonal anti-INCENP (clone P240, Cell Signaling, 2807), rabbit polyclonal anti-pTSS INCENP (a kind gift of M.A. Lampson) [33 (link)], rabbit polyclonal anti-KIF23 (clone N19, Santa Cruz Biotechnology, sc-867), rabbit polyclonal anti-KIF14 (Bethyl Laboratories, A300–233A) and rabbit polyclonal anti-KIF20A (a kind gift of T.U. Mayer) [44 (link)]. Peroxidase and Alexa-fluor conjugated secondary antibodies were purchased from Jackson Laboratories and Invitrogen.

McKenzie C., Bassi Z.I., Debski J., Gottardo M., Callaini G., Dadlez M, & D'Avino P.P. (2016). Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis. Open Biology, 6(3), 160019.

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Western Blotting for Plasmodium Parasites

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Western blotting for Plasmodium parasites was performed as described previously (35 (link), 64 (link)). Briefly, parasites were selectively permeabilized by treatment with ice-cold 0.03% saponin in PBS for 15 min followed by lysis with RIPA to remove the hemozoin. The antibodies used in this study were mouse anti-HA antibody (clone 16B12, BioLegend; 1:500), rabbit polyclonal anti-HA SG77 (ThermoFisher; 1:500), mouse monoclonal anti-FLAG clone M2 (Sigma-Aldrich; 1:200), and mouse monoclonal anti-EXP2 clone 7.7 (1:500). The secondary antibodies used are IRDye 680CW goat anti-mouse IgG (LICOR Biosciences) (1:20,000). Western blot images were processed using the Odyssey Clx LICOR infrared imaging system software (LICOR Biosciences). Full-length blots are presented in Fig. S5.

Fierro M.A., Muheljic A., Sha J., Wohlschlegel J, & Beck J.R. (2024). PEXEL is a proteolytic maturation site for both exported and non-exported Plasmodium proteins. mSphere, 9(2), e00393-23.

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