Anti flag m2 gel

Manufactured by Merck Group

Sourced in United States

The Anti-FLAG M2 gel is a laboratory product used for protein purification. It is designed to bind and purify proteins that are tagged with the FLAG peptide sequence, which is a commonly used affinity tag. The gel consists of an agarose matrix with covalently attached anti-FLAG M2 monoclonal antibodies, which specifically recognize and bind the FLAG tag. This allows for the selective capture and purification of FLAG-tagged proteins from complex mixtures.

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Lab products found in correlation

Protease inhibitor mixture, by Merck Group (2 mentions) Hrp conjugated goat anti mouse or anti rabbit secondary antibody, by Promega (2 mentions) Anti klf4, by Santa Cruz Biotechnology (2 mentions) Ecl reagent, by Bio-Rad (2 mentions) Protein assay reagent, by Bio-Rad (2 mentions) Laemmli sample buffer, by Bio-Rad (2 mentions) Anti ha agarose beads, by Merck Group (1 mentions) Phos 4ebp1, by Cell Signaling Technology (1 mentions) Ha tag, by Proteintech (1 mentions) Azd8055, by MedChemExpress (1 mentions) His tag, by Proteintech (1 mentions) Gapdh, by Proteintech (1 mentions) Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions) Ponceau s, by Avantor (1 mentions) Anti ha hrp antibodies clone 3f10, by Merck Group (1 mentions) Anti flag m2 antibody, by Merck Group (1 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions) Protein a g plus agarose beads, by GE Healthcare (1 mentions) Cellytic m lysis buffer, by Merck Group (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-FLAG (2121 citations) Anti-FLAG M2 affinity gel (1413 citations) Anti-FLAG M2 (1022 citations) FLAG M2 (383 citations) Anti-Flag M2 affinity gel (A2220) (19 citations) Anti-FLAG M2 agarose gel (12 citations) Anti-FLAG M2 affinity gel resin (11 citations) Mouse anti-FLAG M2 affinity gel (9 citations) EZ view red ANTI-FLAG M2 affinity gel beads (9 citations) Anti-FLAG M2 affinity gel agarose beads (5 citations)

15 protocols using anti flag m2 gel

Blimp-1 Posttranslational Modifications Analysis

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For the immunoprecipitation (IP) assay, cells were lysed with 2% SDS lysis buffer (50 mM pH 7.5 Tris and 2% SDS) and boiled for 15–20 min. The lysates were diluted 20-fold in co-IP-lysis buffer 1 (50 mM pH 7.5 Tris, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 2 mM EDTA, freshly added 1 mM PMSF, 20 μM MG132, 10 mM N-ethylmaleimide, 20 mM each protease inhibitor cocktail and phosphatase inhibitor cocktail, and 2 mM DTT), and Blimp-1-flag proteins were immunoprecipitated with anti-FLAG M2 gels (Sigma, Cat: A2220) and analyzed for SUMO-1, SUMO-2/3, or Ub modification.
For LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry) analysis, 293T cells transfected with WT or mutant Blimp-1-flag-expressing plasmids were treated with or without MG132 for 12 h. These cells were lysed with co-IP-lysis buffer 2 (100 mM Tris-HCl, 80 mM NaCl, 1 mM EDTA, 5 mM EGTA, 5% glycerol, 2% w/v digitonin, 0.1% Brij 35, freshly added 1 mM PMSF, 20 μM MG132, 10mM N-ethylmaleimide, 20 mM each protease inhibitor cocktail, and phosphatase inhibitor cocktail), and then proteins were co-immunoprecipitated with anti-FLAG M2 beads and further analyzed by LC-MS/MS.

Wang W.F., Yan L., Liu Z., Liu L.X., Lin J., Liu Z.Y., Chen X.P., Zhang W., Xu Z.Z., Shi T., Li J.M., Zhao Y.L., Meng G., Xia Y., Li J.Y, & Zhu J. (2017). HSP70-Hrd1 axis precludes the oncorepressor potential of N-terminal misfolded Blimp-1s in lymphoma cells. Nature Communications, 8, 363.

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Dephosphorylation of Shootin1a by PP1 and PP2A

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FLAG-shootin1a was expressed in HEK293T cells. To increase phosphorylated FLAG-shootin1a in HEK293T cells, the cells were treated with calyculin A (200 nM) for 1 h. Phosphorylated FLAG-shootin1a was then purified using anti-FLAG M2 gels (Sigma, catalog number: A2220). Recombinant human PP1 alpha and bovine PP2A₂ were purchased from Novus Biologicals (catalog number: NBP1-72418) and Sigma (product number: P1868), respectively. Phosphatase reactions were carried out in PP1 reaction buffer (10 mM Tris-HCl pH 7.0, 50 mM NaCl, 2 mM DTT, 1 mM MnCl₂) or PP2A reaction buffer (50 mM Tris-HCl pH 7.0, 150 mM NaCl, 1 mM DTT, 1 mM MnCl₂) containing 1.5 mU/μl PP1 or 1.6 mU/μl PP2A and 15 ng phosphorylated FLAG-shootin1a. As PP1 at 1.5 mU/μl and PP2 at 1.6 mU/μl dephosphorylated an equivalent amount of the nonpeptide substrate DiFMUP (56 (link)) (Tocris Bioscience, catalog number: 6882) (Fig. S6), the enzymes at these concentrations were used in the shootin1a dephosphorylation assay. The reactions were performed at 30 °C for 3 h. Immunoblot of phosphorylated shootin1a was then performed.

Kastian R.F., Baba K., Kaewkascholkul N., Sasaki H., Watanabe R., Toriyama M, & Inagaki N. (2023). Dephosphorylation of neural wiring protein shootin1 by PP1 phosphatase regulates netrin-1-induced axon guidance. The Journal of Biological Chemistry, 299(5), 104687.

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Co-immunoprecipitation and in vitro Ubiquitination

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Cells were lysed in 1 % Triton X-100 isotonic buffer. Co-immunoprecipitations were performed using anti-V5-agarose gel, anti-HA-agarose beads and anti-Flag-M2 gel (all from Sigma) or 1μg antibody specific for TRIM5, RNF125, RIG-I, TRAF3, TRAF6 or IKKα with UltraLink Protein A/G beads (Pierce). For in vitro ubiquitination assays, mouse BMDC or HEK293 cells were lysed in 1% SDS isotonic buffer. Lysates were boiled for 15 min to remove any non-covalent interactions, followed by centrifugation for 10 min at 15,000 rpm. Supernatants were transferred to a new tube, and diluted with 1% Triton X-100 buffer for immunoprecipitation with anti-Flag M2 or 1μg of anti-RIG-I antibody and UltraLink Protein A/G beads. Immunocomplex was harvested and washed with 0.1 % Triton X-100 isotonic buffer and resuspended with protein loading buffer.

Chen S.T., Chen L., Lin D.S., Chen S.Y., Tsao Y.P., Guo H., Li F.J., Tseng W.T., Tam J.W., Chao C.W., Brickey W.J., Dzhagalov I., Song M.J., Kang H.R., Jung J.U, & Ting J.P. (2019). NLRP12 regulates anti-viral RIG-I activation via interaction with TRIM25. Cell host & microbe, 25(4), 602-616.e7.

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Pharmacological Inhibition of mTOR Pathway

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AZD-8055 was purchased from MedChemExpress (Shanghai, China). Antibodies for western blotting against HPCAL1, ACSS2, RUVBL1, GAPDH, his tag and HA tag were obtained from Proteintech (Wuhan, China). Antibody against phos-mTOR, and phos-4EBP1 and phos-4EBP1 were purchased from Cell Signaling Technology (USA). SCD1 antibody was obtained from ABclonal (Wuhan, China). HPCAL1 antibody for IHC staining and anti-FLAG M2 gel was purchased form Sigma-Aldrich (USA).

Chen T., Yuan Z., Lei Z., Duan J., Xue J., Lu T., Yan G., Zhang L., Liu Y., Li Q, & Zhang Y. (2022). Hippocalcin-Like 1 blunts liver lipid metabolism to suppress tumorigenesis via directly targeting RUVBL1-mTOR signaling. Theranostics, 12(17), 7450-7464.

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Sirt3-mediated deacetylation of mtBER proteins

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Control cells were transfected with Flag-Sirt3 plasmid and test cells with Flag-NEIL1, Flag-NEIL2, Flag-MUTYH, Flag-APE1, and Flag-LIG3 plasmids. Cells were then lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) with the addition of a protease inhibitor at room temperature for 20 minutes and then centrifuged at room temperature. 12,000 xg for 20 minutes. Test proteins were purified using immunopurification (ANTI-FLAG M2 Gel, Sigma). In the deacetylation test, purified mtBER proteins were incubated with the Sirt3-Flag protein in the presence or absence of 1 mM NAD⁺ in deacetylase buffer (50 mM Tris-HCl (pH 9.0), 4 mM MgCl2, 50 mM NaCl, 0.5 mM dithiothreitol) at 30°C for 3 hours, and then the level of acetylated lysine residues in the tested proteins was measured using the ELISA test with Acetyl-Lysine Antibody LS-C71873 (LifeSpan Biosciences) that recognizes Acetylated-Lysine proteins only when posttranslationally modified by acetylation on the epsilon-amino groups of lysine residues. Acetylated lysine level was treated as a measure of deacetylation compared to control.

Kabzinski J., Walczak A, & Majsterek I. (2022). Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer. Genetics Research, 2022, 7299555.

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Co-Immunoprecipitation Experiments with Flag-tagged Proteins

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Co-IP experiments were performed as previously described (Shyian et al., 2020 (link)) with the following modifications. OD₆₀₀ ~ 0.9 cell cultures were used to prepare 1 ml of lysate. Cells were broken with glass beads in a Precellys Evolution homogenizer. 20 µl of anti-Flag M2 gel (Sigma) per 1 ml of lysate were used. Proteins were eluted by boiling at 95 °C for 10 min in 30 µl of 2 x SDS-PAGE buffer (100 mM Tris pH 8, 4% SDS, 10% glycerol, 0.2% bromophenol blue). Total proteins were isolated as previously described (Kushnirov, 2000 (link)). Proteins were separated on 8% PAGE gels, transferred onto Hybond P 0.22 PVDF membrane (GE Healthcare), stained with Ponceau S (Amresco), and blocked in 5% BSA. Anti-HA-HRP antibodies (clone 3F10, Sigma) at 1:5000, anti-FLAG M2 antibodies (Sigma) at 1:5000, goat anti-mouse IgG-HRP (62–6520, Thermo Fisher Scientific) at 1:5000 and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) were used for protein detection.
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).

Malyavko A.N., Petrova O.A., Zvereva M.I., Polshakov V.I, & Dontsova O.A. (2022). Telomere length regulation by Rif1 protein from Hansenula polymorpha. eLife, 11, e75010.

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Immunoprecipitation of CaMKII delta and PKM2

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Cells were extracted with ice-cold CelLytic M lysis buffer (Sigma-Aldrich) plus phosphatase inhibitor cocktail and protease inhibitor cocktail on ice, followed by sonication. Cell extracts were then clarified by centrifugation at 12,000 rpm for 5 minutes, and the supernatants were subjected to immunoprecipitation with mouse anti-CaMKⅡδ monoclonal and rabbit anti-PKM2 polyclonal antibodies at 4°C overnight, followed by additional incubation with Protein A/G PLUS-Agarose beads (GE Healthcare, Uppsala, Sweden) for an additional 3 hours. After washing with ice-cold lysis buffer 3 times, proteins binding to the beads were eluted with 1× sodium dodecyl sulfate loading buffer. For the Flag-tagged proteins, cell extracts were incubated with anti-Flag M2 affinity gel (A2220; Sigma-Aldrich) overnight at 4°C, and the precipitated proteins were eluted following the anti-Flag M2 gel vendor’s recommendation.

Liu J., Tao M., Zhao W., Song Q., Yang X., Li M., Zhang Y., Xiu D, & Zhang Z. (2022). Calcium Channel α2δ1 is Essential for Pancreatic Tumor-Initiating Cells through Sequential Phosphorylation of PKM2. Cellular and Molecular Gastroenterology and Hepatology, 15(2), 373-392.

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Protein Expression Analysis and Immunoprecipitation

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Cells were harvested and lysed in radioimmune precipitation assay lysis buffer (Upstate Biotechnology) containing protease inhibitor mixture (Sigma) or 1x SDS loading buffer. The protein concentration was determined using Bio-Rad protein assay reagent. Western blotting was performed using anti-KLF4 (Santa Cruz Biotechnology), Src (Cell Signaling), p-Src⁴¹⁸ (Cell Signaling), VHL (Cell Signaling), FLAG (Sigma), β-actin (Sigma), p-Tyr (PY20, Santa Cruz Biotechnology), p-Ser/Thr (Clone 22A, BD) and HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (Promega). Signals were detected with ECL reagents (Bio-Rad). Semi-quantification of data was performed using NIH Image J. For immunoprecipitation assay, cell lysate was incubated with anti-FLAG M2 gel (Sigma) or anti-KLF4 (Santa Cruz Biotechnology) antibody overnight at 4 °C on a rotator, followed by addition of protein A/G plus agarose (Pierce) to the reaction containing anti-KLF4 antibody for 2 h at 4 °C. After five washes with radioimmune precipitation assay lysis buffer supplemented with protease inhibitor mixture, complexes were released from the anti-FLAG M2 gel by boiling for 5 min in 2x SDS-PAGE loading buffer.

Zhou Z., Song X., Chi J., Gius D.R., Huang Y., Cristofanilli M, & Wan Y. (2020). Regulation of KLF4 by posttranslational modification circuitry in endocrine resistance. Cellular signalling, 70, 109574.

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Proteomic Analysis of H2O2-Induced NPM1 Modifications

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HEK293T cells transfected with FLAG-NPM1 were treated with 500 μM H₂O₂ for 30 min. FLAG-NPM1 was then immunoprecipitated with anti-FLAG M2 gel (Sigma), mixed with 2 × SDS sample buffer without β-mercaptoethanol and loaded for SDS–PAGE. After examination of the Coomassie Brilliant Blue staining, the gel bands containing FLAG-NPM1 protein were clipped out and cut into small pieces in a 1.5 ml microtube. The chopped gels were washed three times with 50 mM NH₄HCO₃ in 30% acetonitrile by shaking at room temperature (RT) for 20 min. The gels were further incubated with 300 μl of acetonitrile at RT for 10 min. After removing the acetonitrile, the gels were dried and trypsin-digested following the manufacturer's protocol. Peptides were extracted with 85% acetonitrile and 0.1% trifluoroacetic acid for twice. After removing the pieces of gels, the remained solution was concentrated with a SpeedVac concentrator. Then, the peptides were re-dissolved in 30 μl of 0.1% formic acid for liquid chromatography–tandem mass spectrometry analysis (LTQ Orbitrap Elite, Thermo Scientific). The fragment ions observed in the mass spectra were analysed with Mascot.

Yang K., Wang M., Zhao Y., Sun X., Yang Y., Li X., Zhou A., Chu H., Zhou H., Xu J., Wu M., Yang J, & Yi J. (2016). A redox mechanism underlying nucleolar stress sensing by nucleophosmin. Nature Communications, 7, 13599.

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Immunoprecipitation of Rad9 and Neil1

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Anti-FLAG M2-gel (Sigma-Aldrich # A2220) was used for immunoprecipitation (IP) with mES cells ectopically expressing Rad9 proteins, as well as controls, according to the manufacturer's instructions. For IP of Neil1, 500 μg of pre-cleared mES cell lysates were incubated with anti-Neil1 (Abcam # ab21337) or rabbit IgG (Vector Laboratories Inc. # I-1000) antibodies overnight, followed by addition of Protein A/G agarose beads to the mixture. Unbound proteins were separated by centrifugation at 5000X g for 2 min at 4°C, followed by three washes using binding buffer (25 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5 mM DTT and 5% glycerol). Immunoprecipitated proteins were eluted by adding 25 μl of 2X sample buffer (125 mM Tris HCl, pH6.8, 4% SDS, 20% glycerol and 0.004% bromophenol blue) to each reaction. Controls were treated similarly.

Panigrahi S.K., Hopkins K.M, & Lieberman H.B. (2015). Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair. Nucleic Acids Research, 43(9), 4531-4546.

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