Magnetic anti flag m2 beads

Manufactured by Merck Group

Sourced in United States

Magnetic anti-FLAG M2 beads are a type of affinity purification reagent used to isolate and purify proteins or protein complexes that have been tagged with a FLAG epitope. They consist of magnetic beads coated with the anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag. These beads can be used to capture and concentrate FLAG-tagged proteins from complex mixtures, such as cell lysates or culture supernatants, for further analysis or purification.

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M2 magnetic beads FLAG magnetic beads Anti-FLAG M2 Anti-FLAG beads FLAG M2 M2 beads Flag beads Magnetic beads Anti-FLAG

27 protocols using magnetic anti flag m2 beads

Immunoprecipitation of FLAG-tagged Complexes

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Immunoblot analysis and immunoprecipitation were performed as described previously (36 (link)). Briefly, Magnetic M2 anti-FLAG beads (Sigma-Aldrich) were used to immunoprecipitate FLAG-tagged complexes from RNase A-treated (50 μg/ml) HeLa cell lysates in lysis buffer (50 mM Tris [pH 7.2], 150 mM NaCl and 0.5% Triton X-100) supplemented with protease inhibitor (Sigma-Aldrich). Complexes were eluted with SDS-sample buffer, separated by SDS-PAGE and analysed by immunoblotting.

Gromadzka A.M., Steckelberg A.L., Singh K.K., Hofmann K, & Gehring N.H. (2016). A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs. Nucleic Acids Research, 44(5), 2348-2361.

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Immunoprecipitation of FLAG-tagged Complexes

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Twenty‐four hours after transfection, cells were harvested in 400 μl/10‐cm plate of buffer E (20 mM Tris pH 7.5, 150 mM KCl, 0.1% NP40) supplemented with 0.3 mM MgCl₂ and EDTA‐free complete (Sigma‐Aldrich) and lysed 30 min on ice. Magnetic M2 anti‐FLAG beads (Sigma‐Aldrich) were used to immunoprecipitate FLAG‐tagged complexes from RNaseA‐treated (30–40 μg/ml) cell lysates after 1 h incubation with the beads at 4°C. Beads were washed eight times with buffer E supplemented with 0.6 mM MgCl₂. Tubes were changed before the last wash. FLAG complexes were eluted with 25 μl 0.1 M glycine (pH 3.0), added to 6 μl 5× loading buffer and neutralized with 1.5 μl 1 M Tris pH 7.5. 6 μl of the samples was loaded for anti‐FLAG detection and 20 μl for anti‐V5 detection, separated by SDS–PAGE and analysed by immunoblotting using anti‐FLAG and anti‐V5‐antibodies (Sigma‐Aldrich F7425 and V8137, respectively). Anti‐TUBB (Sigma‐Aldrich T5168) and anti‐ACTB (Sigma‐Aldrich A1978) antibodies were used for loading controls.

Neu‐Yilik G., Raimondeau E., Eliseev B., Yeramala L., Amthor B., Deniaud A., Huard K., Kerschgens K., Hentze M.W., Schaffitzel C, & Kulozik A.E. (2017). Dual function of UPF3B in early and late translation termination. The EMBO Journal, 36(20), 2968-2986.

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Immunoprecipitation of FLAG-tagged Proteins

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Membrane fractions were solubilized in buffer (50 mM Tris pH 7.4, 150 mM NaCl, 20 mM MgCl₂, 4 g digitonin/g protein [1% wt/vol], 1× cOmplete protease inhibitor) for 20 minutes and aggregates were pelleted at 20,000g for 20 minutes. Input sample was taken from the supernatant and the rest was incubated with anti-FLAG magnetic M2 beads (Sigma-Aldrich, M8823) for 3 hours. Beads were washed 4 times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 20 mM MgCl₂, 0.1% wt/vol digitonin, 1× cOmplete protease inhibitor). Samples were eluted with Laemmli buffer and analyzed by immunoblotting.

Lone M.A., Aaltonen M.J., Zidell A., Pedro H.F., Morales Saute J.A., Mathew S., Mohassel P., Bönnemann C.G., Shoubridge E.A, & Hornemann T. (2022). SPTLC1 variants associated with ALS produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins. The Journal of Clinical Investigation, 132(18), e161908.

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Membrane Protein Purification Protocol

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Heavy membrane fractions were solubilized in buffer (50 mM TRIS, pH 7.4, 150 mM NaCl, 1% 4 g digitonin [EMD Millipore #300410]/g protein [1% wt/vol], and 1× PI) for 20 min and the aggregates were pelleted at 20,000g for 15 min. The supernatant was incubated with anti-FLAG magnetic M2 beads (M8823; Sigma-Aldrich) for 3 h. For ms analysis, the samples were eluted using 100 mM glycine, pH 2.5, TCA precipitated, and trypsin digest and mass spectrometry analysis was performed at the Institute de Recherches Cliniques de Montreal. For immunoblot analysis samples were eluted with Laemmli buffer. Information for subcellular and sub-mitochondrial localization in Table S2 were retrieved from UniProt Consortium (2021) (link) and MitoCarta3.0 (Rath et al, 2021 (link)), respectively.

Aaltonen M.J., Alecu I., König T., Bennett S.A, & Shoubridge E.A. (2021). Serine palmitoyltransferase assembles at ER–mitochondria contact sites. Life Science Alliance, 5(2), e202101278.

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Co-immunoprecipitation of Pol ι and Myc-tagged proteins

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HEK293T cells were resuspended and sonicated in immunoprecipitation buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, 10 mM MgCl₂, 0.5% Triton X-100) supplemented with 1 × protease inhibitor cocktail, 50 µM PR-619 (Selleckchem) and Pierce Universal Nuclease for Cell Lysis (1:5000, Thermo Fisher Scientific). Where immunoprecipitated proteins were analyzed by mass spectrometry, 0.1% CHAPS was used in place of 0.5% Triton X-100. For immunoprecipitation of Pol ι or Myc-tagged proteins, protein G magnetic Dynabeads (Life Technologies) were prepared by incubation with the Pol ι or Myc antibodies, respectively. For Pol ι co-immunoprecipitation experiments, isotype control IgG-conjugated beads were also prepared by incubating Dynabeads with the Cell Signaling IgG XP isotype control rabbit mAb (clone DA1E, cat # 3900S). Magnetic anti-FLAG M2 beads (Sigma-Aldrich, cat # M8823) were used for the immunoprecipitation of FLAG-tagged proteins. In all cases, conjugated beads were washed in immunoprecipitation buffer, then incubated with whole cell lysates for 1 hour at 4 °C. Beads were then washed 4 × with immunoprecipitation buffer, 2 × with immunoprecipitation buffer modified to contain 250 mM KCl and then proteins eluted by incubating the beads in 100 mM pH 2.3 glycine for 10 min, followed by neutralization of the sample with 500 mM Tris pH 7.4.

Ashton N.W., Valles G.J., Jaiswal N., Bezsonova I, & Woodgate R. (2020). DNA Polymerase ι Interacts with Both the TRAF-like and UBL1-2 Domains of USP7. Journal of molecular biology, 433(2), 166733.

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Immunoprecipitation of Inflammatory Complexes

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MEFs were lysed in 1% Triton-X100 lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X100, 1.25 mM NaF, 0.2 mM NaVO3, 2 mM β-glycerophosphate, 10 mM N-ethylmaleimide and complete TM protease inhibitor cocktail (Roche)], each lysate was normalized by BCA protein assay (Thermo). IKKγ immunoprecipitations were performed using a sheep polyclonal anti-IKKγ antibody at 4 °C for 4 h. Protein G Dynabeads (Thermo) were added for the final 1 h of incubation. Active TNFR complex IPs were performed with magnetic anti-Flag (M2) beads (Sigma) by incubation at 4 °C for 2 h. IL-1β IPs were performed by incubating lysates with either biotinylated anti-IL-1β (BioLegend B122) or biotinylated IgG1 isotype control for 16 h followed by adding Neutravidin Agarose (Pierce) for 1 h. Beads were then washed three times and samples were eluted from the beads using 2X LDS buffer (Thermo) containing 715 mM β-mercaptoethanol.

Razani B., Whang M.I., Kim F.S., Nakamura M.C., Sun X., Advincula R., Turnbaugh J.A., Pendse M., Tanbun P., Achacoso P., Turnbaugh P.J., Malynn B.A, & Ma A. (2020). Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis-like disease and inflammation. Nature immunology, 21(4), 422-433.

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Immunoprecipitation of FLAG-tagged proteins

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Cells were resuspended in immunoprecipitation buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5% glycerol, 10 mM MgCl₂, 0.5% Triton X-100) supplemented with 1× phosphatase inhibitor cocktail (CST, cat # 5870), 1× protease inhibitor cocktail (Sigma-Aldrich, cat # 11697498001) and universal nuclease for cell lysis (1:2000, ThermoFisher Scientific) and lysed by sonication (Vibra-Cell, 3 mm probe; Sonics and Materials) with 3 × 3 s bursts (10% output). For the immunoprecipitation of FLAG-tagged proteins, 1000 μg of whole cell lysate was incubated with magnetic anti-FLAG M2 beads (Sigma-Aldrich) for 1 h at 4°C, beads washed five times in immunoprecipitation buffer and protein eluted by heating to 80°C for 5 min in 3× SDS loading dye. Eluted proteins were then immunoblotted as per above.

Touma C., Adams M.N., Ashton N.W., Mizzi M., El-Kamand S., Richard D.J., Cubeddu L, & Gamsjaeger R. (2017). A data-driven structural model of hSSB1 (NABP2/OBFC2B) self-oligomerization. Nucleic Acids Research, 45(14), 8609-8620.

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Purification of Yta7 phosphorylation by CDK

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One hundred nanomolar of FLAG-tagged Yta7, as well as mutant versions were incubated with 25 nM S-CDK (Clb5-Cdc28) in buffer containing 100 mM K-glutamate, 25 mM HEPES-KOH pH 7.6, 10 mM Mg(OAc)₂, 0.02% NP-40, 1 mM DTT, 0.1 mg/mL BSA and 5 mM ATP for 30 min at 30 °C. Sic1 (250 nM) was then added and reactions were incubated for another 3 min at 30 °C. Samples were then diluted in the same buffer (100 mM K-glutamate) without ATP and incubated with magnetic anti-FLAG M2 beads (Sigma, M8823) for 45 min at 4 °C with rotation. Beads were then washed 6 times with buffer as above plus 300 mM K-glutamate and without ATP and once with buffer containing 150 mM KCl, 25 mM Tris-HCl pH 7.2 (25 °C), 10% glycerol, 0.05% NP-40, 1 mM EDTA, and 4 mM MgCl₂. Proteins were eluted in the same buffer supplemented with 0.25 mg/mL 3× FLAG peptide with shaking for 1 h at 4 °C. The supernatant was collected, aliquoted, and flash-frozen in liquid nitrogen for −80 °C storage. Protein concentrations were determined using a Bradford reagent (Biorad) and equal protein amounts were additionally verified by silver staining (Invitrogen).

Chacin E., Bansal P., Reusswig K.U., Diaz-Santin L.M., Ortega P., Vizjak P., Gómez-González B., Müller-Planitz F., Aguilera A., Pfander B., Cheung A.C, & Kurat C.F. (2021). A CDK-regulated chromatin segregase promoting chromosome replication. Nature Communications, 12, 5224.

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Investigating SPRTN Interactome

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To test binding between USP7/VCPIP1/USP11 and SPRTN, HeLa-T-REx Flp-In cells stably expressing YFP-SPRTN-Strep variants were seeded in 60 mm tissue culture plates, grown to 50% confluency and then transiently transfected with pcDNA5-FRT/TO plasmids encoding Flag/Flag-USP7/VCPIP1/USP11 variants using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Sixteen hours after transfection and concurrent induction of protein expression by doxycycline (1 μg/ml), cells were washed once with ice-cold PBS and harvested by scraping in lysis buffer (20 mM Tris/HCl pH 7.5, 137 mM NaCl, 1% IGEPAL CA-630, 2 mM EDTA, 2 mM MgCl₂, 4 U/ml Benzonase, cOmplete EDTA free protease inhibitors, 0.04 mg/ml PefaBloc SC and 20 mM iodoacetamide). Lysates were incubated for 30 min on ice, before centrifugation at 16 000 g for 30 min. Supernatants were then used for immuno-precipitation using 5 μl magnetic anti-Flag M2 beads (Sigma) at 4°C for 1 h. The beads were then washed three times with wash buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA and 0.5% IGEPAL CA-630). Finally, samples were resuspended in 100 μl 1× LDS sample buffer, analysed by SDS-PAGE and western blotting with anti-Flag, anti-GAPDH and anti-Strep antibodies.

Zhao S., Kieser A., Li H.Y., Reinking H.K., Weickert P., Euteneuer S., Yaneva D., Acampora A.C., Götz M.J., Feederle R, & Stingele J. (2020). A ubiquitin switch controls autocatalytic inactivation of the DNA–protein crosslink repair protease SPRTN. Nucleic Acids Research, 49(2), 902-915.

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Investigating mArnt2 Regulation and Function

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HEK293T cells (ATCC) were cultured in high-glucose DMEM (Gibco Life Technologies) containing 10% (vol/vol) FBS (HyClone), 10,000 units/ml penicillin and 10,000 μg/ml streptomycin at 37°C. Cells were tested periodically for mycoplasma contamination by Cell Culture Contamination Detection Kit (Thermo Fisher Scientific). Murine Arnt2 (mArnt2) cDNA, HA-Sim1 cDNA, and 6×CME and mutant 6×CME luciferase were a kind gift from J. Pelletier (Moffett and Pelletier, 2000 (link)). mArnt2 was cloned into a FLAG-tagged expression vector (Clontech). FLAG was immunoprecipitated using magnetic anti-FLAG M2 beads (Sigma). Nuclear and cytosolic lysates were generated using a Cell Fractionation kit (Cell Signaling Technology) as per the manufacturer’s guidelines. The Dual-Glo Luciferase Assay System was performed per assay instructions (Promega). Antibodies for western blot were commercially obtained: anti-FLAG M2 (Sigma, F1804; 1:2000); anti-ARNT2 (Abcam, ab70122; 1:1000); anti-GAPDH (Cell Signaling Technology, 5174S; 1:1000) and anti-histone H3 (Cell Signaling Technology, 9715S; 1:1000).

Turer E.E., San Miguel M., Wang K.W., McAlpine W., Ou F., Li X., Tang M., Zang Z., Wang J., Hayse B., Evers B., Zhan X., Russell J, & Beutler B. (2018). A viable hypomorphic Arnt2 mutation causes hyperphagic obesity, diabetes and hepatic steatosis. Disease Models & Mechanisms, 11(12), dmm035451.

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