Flag magnetic beads

Manufactured by Merck Group

Sourced in United States

FLAG magnetic beads are a type of laboratory equipment used for protein purification and immunoprecipitation. They consist of magnetic particles coated with an anti-FLAG antibody, which allows for the selective capture and isolation of proteins tagged with a FLAG epitope.

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Lab products found in correlation

Flag peptide, by Merck Group (2 mentions) Protein a magnetic beads, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Tab edta, by Roche (2 mentions) Protease inhibitor, by Roche (2 mentions) Antibodies against β actin, by Santa Cruz Biotechnology (1 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions) Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions) Dual luciferase reporter assay system, by GenePharma (1 mentions) Phosphatase inhibitors cocktail, by Roche (1 mentions) Bca protein assay kit, by Solarbio (1 mentions) Rabbit anti flag antibody, by Merck Group (1 mentions) Anti myc, by Thermo Fisher Scientific (1 mentions) Anti ty1, by Diagenode (1 mentions) 3xflag peptide, by Apexbio (1 mentions) Benzonase, by Merck Group (1 mentions) Ab8224, by Abcam (1 mentions) Ab14705, by Abcam (1 mentions) Ab14734, by Abcam (1 mentions) Ab117991, by Abcam (1 mentions)

Close spelling variants of this product

Anti-FLAG M2 magnetics beads Anti-FLAG M2 magnetic beads Anti-FLAG M2 magnetic bead slurry Anti-FLAG-coated magnetic beads α-FLAG M2 magnetic beads Anti-flag M2 Magnetic Beads (M8823), Anti-FLAG M2 magnetic beads for FLAG pulldown α-FLAG magnetic beads Anti-FLAG antibody-conjugated magnetic beads M2 Flag conjugated magnetic beads

28 protocols using flag magnetic beads

Affinity Purification and Mass Spectrometry of TEM8 Interactors

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Protein lysates extracted from EBC buffer (50 mM Tris, 120 mM NaCl, 0.5% NP40, pH7.5) were incubated with magnetic FLAG-Beads (Sigma-Aldrich) or anti-HA antibody-conjugated protein A/G agarose beads (Thermo Fisher) overnight at 4 °C. After washing with NETN buffer (20 mM Tris, 100 mM NaCl, 0.5% NP40, 1 mM EDTA, pH8.0), the complexes were eluted to follow Western blotting.
For mass spectrometry analysis, cells (MDA-MB-231-ctrl, MDA-MB-231-TEM8, SUM159-ctrl, and SUM159-TEM8) were treated with MG132 (20 uM, 2 h) before being harvested. IP samples (on FLAG-beads) were dissolved with 50 mM ammonium bicarbonate solution and digested by trypsin at 37 °C for 16 h. After being centrifuged, the supernatant was freeze-dried and desalted, followed by adding 0.1% formic acid and vortexed to be fully dissolved. The samples were then centrifuged and the supernatant was added to the sample bottle for mass spectrometry (Orbitrap Elite) detection. Search database was Maxquant. In the initial screening, proteins with high LFQ intensity (>10⁶) in TEM8-overexressing groups but not in ctrl groups (LFQ intensity is 0) were picked out and identified as possible TEM8-interacting proteins. Antibody information used in western blotting and IP were listed in Supplementary Table 5.

Xu J., Yang X., Deng Q., Yang C., Wang D., Jiang G., Yao X., He X., Ding J., Qiang J., Tu J., zhang R., Lei Q.Y., Shao Z.M., Bian X., Hu R., Zhang L, & Liu S. (2021). TEM8 marks neovasculogenic tumor-initiating cells in triple-negative breast cancer. Nature Communications, 12, 4413.

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Chromatin Immunoprecipitation of FLAG-tagged ERα

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In total, 293 T cells were transfected with a pSIN vector containing a FLAG-tagged ERα fragment and cultured for two days. The magnetic FLAG-beads (Sigma-Aldrich) were used to pull down the ERα protein and binding DNA. A nonspecific normal IgG was used as negative control. And the purified DNA was used to perform subsequent qRT-PCR detection. We designed two pairs of primers to target different regions of the ASB10 promoter, as listed in Supplementary Table 4.

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Investigating BYSL and RIOK2 Interaction

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A rabbit anti-BYSL polyclonal antibody from Sigma-Aldrich (St. Louis, MO, USA) was used for Western blot (1:500) and immunohistochemistry (1:200) experiments. A rabbit anti-RIOK2 polyclonal antibody (1:50; Abnova, Taibei City, Taiwan) and a mouse anti-RIOK2 polyclonal antibody (1:400; Sigma-Aldrich) was used for Western blot and immunofluorescence, respectively. Magnetic FLAG beads (Sigma-Aldrich), Protein A/G PLUS-Agarose, and normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in the immunoprecipitation assays. Antibodies against β-actin (1:1,500; Santa Cruz Biotechnology), FLAG (1:1,000; Sigma-Aldrich), Myc, and the signaling molecules (1:1,000; Cell Signaling Technology, Danvers, MA, USA) were used for Western blot analysis. The Myc-tagged RIOK2-overexpressing plasmid was obtained from the Chinese Science Academy (Beijing, China)^{14 (link)}, and the FLAG-tagged BYSL-overexpressing plasmid was purchased from Viogene Biosciences (Jinan, China).

Gao S., Sha Z., Zhou J., Wu Y., Song Y., Li C., Liu X., Zhang T, & Yu R. (2021). BYSL contributes to tumor growth by cooperating with the mTORC2 complex in gliomas. Cancer Biology & Medicine, 18(1), 88-104.

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Protein Immunoprecipitation and Mass Spectrometry

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Cells were lysed with EBC buffer (50 mmol/L Tris, 120 mmol/L NaCl, 0.5% NP40, pH 7.5) to obtain a protein lysate. Magnetic FLAG-Beads (Sigma-Aldrich) or anti-HIS antibody-coupled protein A/G agarose Beads (Thermo Fisher) were added to the protein lysate, and samples were rotated overnight at 4°C. After washing with NETN buffer (20 mmol/L Tris, 100 mmol/L NaCl, 0.5% NP40, 1 mmol/L EDTA, pH 8.0), the complexes were eluted and subjected to Western blot. For mass spectrometry analysis, cells were pretreated with MG132 (10 μmol/L, 2 h) before being harvested. Samples were separated by SDS-PAGE electrophoresis, dissolved with 50 mmol/L ammonium bicarbonate solution, and trypsinized at 37°C for 20 h. After centrifugation, the supernatant was lyophilized, desalted, and vortexed to dissolve by adding 0.1% formic acid. The samples were then centrifuged, and the supernatant was added to vials for detection by mass spectrometry (Orbitrap Elite). The search database was Maxquant. Antibody information used for Western blot and Immunoprecipitation is listed in Table S5.

Ma W., Zhang L., Chen W., Chang Z., Tu J., Qin Y., Yao Y., Dong M., Ding J., Li S., Li F., Deng Q., Yang Y., Feng T., Zhang F., Shao X., He X., Zhang L., Hu G., Liu Q., Jiang Y.Z., Zhu S., Xiao Z., Su D., Liu T, & Liu S. (2024). Microbiota enterotoxigenic Bacteroides fragilis-secreted BFT-1 promotes breast cancer cell stemness and chemoresistance through its functional receptor NOD1. Protein & Cell, 15(6), 419-440.

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Arl3-FLAG Immunoprecipitation Protocol

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10-cm confluent plates of transiently transfected AD-293 cells were washed well with PBS and then incubated in 1 mM disuccinimidyl suberate (Thermo Scientific, 21555) in PBS at 37°C for 10 min. The reaction was quenched by adding Tris Buffer pH 8, to a final concentration of 100 mM for 15 min at room temperature. After washing again with PBS, cells were collected and lysed in 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.5% Igepal, 2% glycerol with cOmplete protease inhibitor cocktail. Immunoprecipitations were performed by adding 15 µl of magnetic FLAG beads (Sigma-Aldrich, M8823) to the lysates and rotating at 4°C for 2 hr before washing three times with a high salt buffer (50 mM Tris pH 7.5, 500 mM NaCl, 5 mM MgCl₂, 2% glycerol) and then eluting by shaking with 15 µl of 100 µg/µl of 3× FLAG peptide (Sigma-Aldrich, F4799) in the original lysis buffer for 30 min at 4°C. A minimum of three replicates was performed for each Arl3-FLAG construct and amount of Arl13B-GFP binding to each Arl3-FLAG is shown in Figure 3—figure supplement 3.

Travis A.M., Manocha S., Willer J.R., Wessler T.S., Skiba N.P, & Pearring J.N. (2023). Disrupting the ciliary gradient of active Arl3 affects rod photoreceptor nuclear migration. eLife, 12, e80533.

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Detailed Biochemical Assays for Liver Tissue

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Liver tissue was pretreated with NP-40 lysate, the samples were dissolved in assay buffer and vortexed extensively for 2 min as described previously [27 (link)], and then, TG content was determined according to the manufacturer’s instruction (Abcam, Cambridge, UK). The luciferase activity assay was carried out using the Dual-Luciferase Reporter Assay System (GenePharma, Shanghai, China). HEK293T cells were cotransfected with miR-743a-3p mimic/inhibitor and GSTM1-3′-UTR reporter. Luciferase activity was evaluated 48 h after transfection, as described previously [28 (link)]. The coimmunoprecipitation (co-IP) assay was performed as Sun, et al. described [29 (link)]. In brief, cells were lysed in co-IP buffer on ice for 30 min. Then, the cells were centrifuged, and the supernatant was collected, followed by incubation with Flag Magnetic Beads (Sigma-Aldrich, St. Louis, MO) with gentle rocking overnight at 4 °C. The mixture pelleted was washed three times and then eluted in a loaded buffer and denatured at 95 °C for 10 min before western blotting. The reagents and methods for malonaldehyde (MDA), GST activity, mRNA and protein expression, immunohistochemistry, and immunofluorescence are detailed in Additional file 1.

Xu T., Pan Y., Ding Q., Cao F., Chang K., Qiu J., Zhuge H., Hao L., Wei H., Si C., Dou X, & Li S. (2024). The micro-743a-3p–GSTM1 pathway is an endogenous protective mechanism against alcohol-related liver disease in mice. Cellular & Molecular Biology Letters, 29, 35.

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Isolation of HO-1 Protein Complexes

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PC3 cells were transfected either with FLAGHO-1 plasmid or the empty vector as a control. Forty-eight hours after transfection, proteins were extracted using a buffer with a low NaCl concentration (20 mM Tris, 150 mM NaCl, 5 mM MgCl₂, 0.5% NP40, pH 7,5) not to disrupt the protein–protein interactions. Protein extracts were incubated with Flag Magnetic Beads (purchased from Sigma-Aldrich, UK) for 2 h at 4 °C. After removing the proteins that did not interact with the FLAG construction, the proteins complexes formed were incubated with 3XFLAG peptide (100 μg/μl) for 2 h at 4 °C. Flag peptide competes with the proteins complexes bound to the magnetic beads and, as a consequence, the FLAGHO-1-interacting proteins remain in the supernatant.

Paez A.V., Pallavicini C., Schuster F., Valacco M.P., Giudice J., Ortiz E.G., Anselmino N., Labanca E., Binaghi M., Salierno M., Martí M.A., Cotignola J.H., Woloszynska-Read A., Bruno L., Levi V., Navone N., Vazquez E.S, & Gueron G. (2016). Heme oxygenase-1 in the forefront of a multi-molecular network that governs cell–cell contacts and filopodia-induced zippering in prostate cancer. Cell Death & Disease, 7(12), e2570-.

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Co-immunoprecipitation Workflow for Protein Interactions

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Cells were washed twice with cold DPBS (#CC010, Macgene) and then lysed with TENT 1% (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, pH 7.4) on ice in the presence of protease inhibitors (#4693132001, Roche) and phosphatase inhibitors cocktail (#04906837001, Roche). Cell lysates were centrifuged at 13, 500g for 5 min at 4 °C to remove debris. Protein concentrations were determined with the BCA Protein Assay kit (#PC0020, Solarbio). Equal amounts of total protein from lysates were immunoprecipitated with FLAG magnetic beads (#M8823, Sigma-Aldrich), HA magnetic beads (#88837, Pierce), or rabbit polyclonal antibody with Protein G agarose (#20398, Pierce) and then eluted with SDS-PAGE loading buffer. Co-immunoprecipitations of PP5 and SGK1 were carried out with Strep-Tactin XT Superflow (#2-4010-010, Neuromics) and eluted with buffer BXT (0.1 M Tris–Cl, 0.15 M NaCl, 1 mM EDTA, 50 mM Biotin (#6-6325-001, IBA), pH 8.0) or with HA magnetic beads.

Gu W., Zheng H, & Canessa C.M. (2023). Phosphatases maintain low catalytic activity of SGK1: DNA damage resets the balance in favor of phosphorylation. The Journal of Biological Chemistry, 299(8), 104941.

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Identifying Protein Interactors via Co-IP

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Co-immunoprecipitation (co-IP) was performed based on a standard protocol [34 (link)] using Flag magnetic beads (M8823, Sigma, Saint Louis, MO, USA) and HA affinity agarose beads (E6779, Sigma, Saint Louis, MO, USA), and the immunoprecipitans were measured by Western blotting. Mass spectrometry analysis was used to screen CLU-interactive proteins from individual bands after the eluted proteins were separated on SDS–PAGE gels and stained with Coomassie blue.

Huang S., Li X., Gu W., Li X., Zhao J., Wu J., Cai J., Feng X, & Tao T. (2022). Cytoplasmic Clusterin Suppresses Lung Cancer Metastasis by Inhibiting the ROCK1-ERK Axis. Cancers, 14(10), 2463.

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Protein Interaction Profiling in HEK293 Cells

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PHF7^Flag-HA was co-expressed with GFP, Gata4^Myc, Hand2^Myc or Mef2c^Myc, or SMARCD3^Flag-HA was co-expressed with PHF^3xTy1 or GFP in HEK293 cells for 72 h. Experiments involving crosslinking were adapted from Zlatic et al.³⁷, whereby cells were treated with 200 μM dithiobis(succinimid ylpropionate) (DSP) or dimethylsulfoxide (DMSO) for 4 h at 4 °C and quenched with 25 mM Tris for 15 min. Pre-cleared lysates were incubated with Flag magnetic beads (Sigma, M8823) overnight. The Flag epitope tag was eluted using 0.5 mg ml⁻¹ free 3× Flag peptide (Sigma). The final elution and input obtained before immunoprecipitation were analysed by western blotting using an anti-Myc (Invitrogen), anti-Ty1 (Diagenode) or rabbit anti-Flag antibody (Sigma).

Garry G.A., Bezprozvannaya S., Chen K., Zhou H., Hashimoto H., Morales M.G., Liu N., Bassel-Duby R, & Olson E.N. (2021). The histone reader PHF7 cooperates with the SWI/SNF complex at cardiac super enhancers to promote direct reprogramming. Nature cell biology, 23(5), 467-475.

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