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The Flag M2 is a recombinant antibody that recognizes the Flag epitope tag. It is commonly used in various applications for the detection and purification of proteins tagged with the Flag epitope.

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Lab products found in correlation

β actin, by Merck Group (2 mentions) Turbofect, by Thermo Fisher Scientific (1 mentions) V5 epitope tag, by Cell Signaling Technology (1 mentions) Ubiquitin, by Santa Cruz Biotechnology (1 mentions) Sharpin, by Proteintech (1 mentions) Hoil 1, by Santa Cruz Biotechnology (1 mentions) Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) C2 confocal microscope, by Nikon (1 mentions) Fibronectin, by BD (1 mentions) Nis software, by Nikon (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions) Valinomycin, by Merck Group (1 mentions) Monensin, by Merck Group (1 mentions) Hdac6, by Cell Signaling Technology (1 mentions) Ouabain, by Merck Group (1 mentions) Gramicidin a, by Merck Group (1 mentions) Psuper basic, by Oligoengine (1 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)

5 protocols using flag m2

1

Cell Line Maintenance and Transduction

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HEK293T, A172, U87 and SKNSH cells were maintained in Eagle’s MEM (U87, SKNSH), DMEM (A172, HEK293T), or L-15 (SW620) media (Invitrogen), all with 10% Fetal Bovine Serum (Hyclone). Cells were transfected with PEI or Turbofect (Fisher) according to the manufacturer’s instructions. Protein expression was analyzed by western blot with antibodies to the Flag (Sigma, Flag M2) or V5 epitope tag (Cell Signaling, 13202). Expression of YAP1 was monitored using a YAP/TAZ specific antibody from Santa Cruz (SC-101119), and HSP90 was detected with an antibody from Cell Signaling (#4874). U87 and A172 cells were transduced with lentiviral vectors under standard conditions using polybrene, and 72 hours after infection cells were subjected to selection with 1μg/ml Puromycin or 2μg/ml Blasticidin, or 0.5μg/ml Puromycin plus 2μg/ml Blasticidin. Assays on infected cells were performed after at least 10 days in selection. A172 and U87 cells were authenticated by STR profiling, in accordance with ICLAC guidelines, at the University of Arizona Genetics Core.
Melhuish T.A., Kowalczyk I., Manukyan A., Zhang Y., Shah A., Abounader R, & Wotton D. (2018). Myt1 and Myt1l transcription factors limit proliferation in GBM cells by repressing YAP1 expression.. Biochimica et biophysica acta. Gene regulatory mechanisms, 1861(11), 983-995.
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2

Immunoblotting Analysis of Innate Immune Signaling

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Proteins were separated by SDS-PAGE or SDD-AGE and transferred to PVDF membrane, blocked with 5% non-fat dry milk in TBS containing 0.1% Tween 20. Membranes were incubated with the primary antibodies against: HOIL1 (Santa Cruz Biotechnologies, sc393754), HOIP (Abcam, ab46322), SHARPIN (Proteintech, 14626-I-AP), FLAG (M2, Sigma), MAVS (Cell Signaling Technologies (CST), 4983S), MDA5 (Adipogen, AL180), LGP2 (Proteintech, 11355–1-AP), IRF3 (CST, 11904S), IRF3-pS396 (CST, 29047S), cytochrome c (CST, 11940S), HA (CST, 3724S), V5 (CST, 13202S), ubiquitin (Santa Cruz Biotechnologies, sc-8017), M1-ubiquitin (Sigma-Aldrich, MABS451), β-actin (Sigma), GAPDH (CST, 97166S), followed by incubation with goat anti-rabbit or anti-mouse IgG-HRP. Blots were incubated with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and imaged on a ChemiDoc Imaging System (Bio-Rad).
Cheng D., Zhu J., Liu G., Gack M.U, & MacDuff D.A. (2024). HOIL1 mediates MDA5 activation through ubiquitination of LGP2. bioRxiv.
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3

Immunofluorescent Labeling of Protein Colocalization

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Cells were seeded onto glass coverslips pre-coated with fibronectin (10 μg/ml; BD Bioscience, San Jose, CA, USA) and allowed to attach overnight. The next day, cells were fixed with ice-cold methanol and stained with the following primary antibodies: FLAG (M2; 1:300 dilution; Cell Signaling Technology), HA (16B12; 1:300 dilution; Covance), and CD98hc (H-300; 1:200 dilution; Santa Cruz Biotechnology). Immunofluorescent images were acquired at room temperature using the C2+ confocal microscope (Nikon) with a normal PMT (Nikon) and a CFI Apochromat Lambda S 60 × NA1.49 oil immersion objective (Nikon) after excitation with 405, 488, and 561 nm laser lines. Confocal images were analyzed using NIS software (Nikon) to obtain Pearson’s correlation.
Kim J.E., Kim H.J., Jung J.W., Song D.G., Park D., Lee H., Um H., Park J., Nam S.H., Cho M, & Lee J.W. (2019). TM4SF5-mediated CD44v8-10 splicing variant promotes survival of type II alveolar epithelial cells during idiopathic pulmonary fibrosis. Cell Death & Disease, 10(9), 645.
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4

Investigating Protein-Protein Interactions

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Co-immunoprecipitation was performed using cell lysates from LNCaP and 293T cells for endogenous and ectopically expressed proteins, respectively, for investigating protein–protein interaction has been described [7 (link)]. The immunoblotting assay was performed using the following antibodies: Flag-M2, Myc-tag (Cell Signaling), KDM8 [17 (link)]; β-actin (Sigma-Aldrich); PSA (Santa Cruz Biotech); ANCCA [61 (link)], EZH2 (Cell signaling), and GAPDH (Santa Cruz Biotech).
Wang H.J., Pochampalli M., Wang L.Y., Zou J.X., Li P.S., Hsu S.C., Wang B.J., Huang S.H., Yang P., Yang J.C., Chu C.Y., Hsieh C.L., Sung S.Y., Li C.F., Tepper C.G., Ann D.K., Gao A.C., Evans C.P., Izumiya Y., Chuu C.P., Wang W.C., Chen H.W, & Kung H.J. (2018). KDM8/JMJD5 as a dual coactivator of AR and PKM2 integrates AR/EZH2 network and tumor metabolism in CRPC. Oncogene, 38(1), 17-32.
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5

Molecular Pathway Modulation Assay

Cited 1 time
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Ouabain, gramicidin A, valinomycin, monensin, PP2, PD98059, LY294002 and BAPTA-AM were obtained from Sigma (St. Louis, MO) and EGF was from Invitrogen. Isotonic sodium buffer was composed of 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM Glucose and 20 mM HEPES, pH 7.4. For low-sodium buffer NaCl was replaced with RbCl [21 (link)]. Antibodies recognizing EGFR, phospho-EGFR (Y1173), acetyl-α-tubulin (K40), α-tubulin, phospho-Akt (S473), phospho-Erk (T202/Y204), GAPDH, HDAC6, Flag (M2), and EEA1, were purchased from Cell Signaling (Beverly, MA), anti-LAMP2 was obtained from Developmental Studies Hybridoma Bank, University of Iowa.
Flag-tagged HDAC6 cDNA was obtained from Addgene (Cambridge, MA) and transfected into human medulloblastoma DAOY cells (ATCC, Rockville, MD) using Lipofectamine 2000 (Invitrogen). For stable knockdown, shRNA targeting human HDAC6 was inserted into pSUPER.basic (Oligoengine, Seattle, WA). Stable clones were selected after treatment with G418. Control clones were obtained in parallel after transfection with pSUPER vector.
Lee S.J., Li Z., Litan A., Yoo S, & Langhans S.A. (2015). EGF-induced sodium influx regulates EGFR trafficking through HDAC6 and tubulin acetylation. BMC Cell Biology, 16, 24.
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