M2 magnetic beads

Manufactured by Merck Group

M2 magnetic beads are a type of laboratory equipment used for a variety of applications. They are small, uniform paramagnetic particles that can be easily separated from a solution using a magnetic field. The core function of M2 magnetic beads is to provide a simple and effective way to capture, isolate, and purify target molecules such as proteins, nucleic acids, or cells, enabling researchers to perform various downstream analyses and experiments.

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Lab products found in correlation

Gfp trap magnetic beads, by Proteintech (2 mentions) Rnase a, by Thermo Fisher Scientific (2 mentions) Proteinase inhibitor cocktail, by Roche (2 mentions) Magna rip kit, by Merck Group (1 mentions) Transfer unit, by Bio-Rad (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Atp γ s, by Abcam (1 mentions) P nitrobenzyl mesylate, by Abcam (1 mentions) Protein g a garose beads, by Yeasen (1 mentions) Anti smurf2, by Cell Signaling Technology (1 mentions) Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-FLAG M2 magnetic beads (753 citations) Magnetic beads (109 citations) FLAG M2 magnetic beads (89 citations) M2 beads (52 citations) Magnetic anti-FLAG M2 beads (27 citations) Anti-FLAG M2 Magnetic Agarose Beads (7 citations) Anti-flag M2 Magnetic Beads (M8823), (6 citations) Anti-FLAG M2 magnetic bead slurry (5 citations) Mouse anti-FLAG M2 magnetic beads (5 citations) α-FLAG M2 magnetic beads (4 citations)

10 protocols using m2 magnetic beads

Immunoaffinity Purification of Protein Complexes

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Cells were washed twice with PBS and lysed with IP lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 10% glycerol) supplemented with proteinase inhibitor cocktail (Roche; 1183617001). Lysates were centrifuged at 4°C for 15 min at 20,000 × g. Supernatants were incubated with anti-FLAG M2 magnetic Beads (Millipore; M8823) or GFP-Trap magnetic beads (ChromoTek; gtma10) at 4°C overnight. Beads were washed three times with lysis buffer, with the second wash supplemented with 0.1 mg/mL RNaseA (Thermo Fisher; EN0531) to remove RNA and hence the RNA-mediated protein-protein interactions. The beads were boiled in 1× NuPAGE LDS sample buffer at 70°C for 5 min and analyzed by immunoblotting.

Yang Z., Johnson B.A., Meliopoulos V.A., Ju X., Zhang P., Hughes M.P., Wu J., Koreski K.P., Clary J.E., Chang T.C., Wu G., Hixon J., Duffner J., Wong K., Lemieux R., Lokugamage K.G., Alvarado R.E., Crocquet-Valdes P.A., Walker D.H., Plante K.S., Plante J.A., Weaver S.C., Kim H.J., Meyers R., Schultz-Cherry S., Ding Q., Menachery V.D, & Taylor J.P. (2024). Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity. Cell reports, 43(3), 113965.

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Protein Immunoprecipitation and Analysis

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Cells were washed twice with PBS and lysed with IP lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 10% glycerol) supplemented with proteinase inhibitor cocktail (Roche; 1183617001). Lysates were centrifuged at 4°C for 15 min at 20,000 x g. Supernatants were incubated with anti-FLAG M2 magnetic Beads (Millipore; M8823) or GFP-Trap magnetic beads (ChromoTek; gtma-10) at 4°C overnight. Beads were washed three times with lysis buffer, with the second wash supplemented with 0.1 mg/ml RNaseA (Thermo Fisher; EN0531) to remove RNA and hence the RNA-mediated protein-protein interactions. The beads were boiled in 1× NuPAGE LDS sample buffer at 70°C for 5 min and analyzed by immunoblotting.

Yang Z., Johnson B.A., Meliopoulos V.A., Ju X., Zhang P., Hughes M.P., Wu J., Koreski K.P., Chang T.C., Wu G., Hixon J., Duffner J., Wong K., Lemieux R., Lokugamage K.G., Alvardo R.E., Crocquet-Valdes P.A., Walker D.H., Plante K.S., Plante J.A., Weaver S.C., Kim H.J., Meyers R., Schultz-Cherry S., Ding Q., Menachery V.D, & Taylor J.P. (2023). Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication. bioRxiv.

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Affinity Purification of HuR-Bound RNAs

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Magna RIP Kit (Merck Millipore, CAT#17-700) was used, but the beads in the kit were replaced with M2 magnetic beads (Sigma-Aldrich), to pull down the proteins with Flag-tagged HuR. Cells were seeded in 10-cm plates and washed twice with 5 ml ice-cold PBS. Cell pellet was collected and resuspended in complete RIP lysis buffer. After using RIP wash buffer to wash 50 µl of M2 magnetic beads twice, 100 µl of lysis and beads were incubated in 900 µl of RIP immunoprecipitation buffer at 4°C overnight. Then, beads were washed by RIP wash buffer five times and collected by a magnetic separator. The immunoprecipitate was resuspended in 150 µl proteinase K buffer and incubated at 55°C for 30 min. The supernatant was transferred into 250 µl of RIP wash buffer and 400 µl of phenol:isoamyl alcohol in the tube. Then, the aqueous phase was moved into a new tube and mixed with 400 µl chloroform. Salt solution was added to enhance the precipitation of RNA at −80°C overnight. Finally, the pellet was washed by 80% ethanol and resuspended in 15 µl of RNase-free water.

Han F., Yang B., Zhou M., Huang Q., Mai M., Huang Z., Lai M., Xu E, & Zhang H. (2022). GLTSCR1 coordinates alternative splicing and transcription elongation of ZO1 to regulate colorectal cancer progression. Journal of Molecular Cell Biology, 14(2), mjac009.

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Protein-DNA Binding Assay for GLTSCR1

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PCDNA3.1 + Flag-tagged GLTSCR1 vector and empty vector were overexpressed in 293 T, and proteins were enriched by M2 magnetic beads (Sigma, CAT#M8823). Purified proteins and biotin-labeled DNA (Motif probe F: TTTAAAATAAAAATT; Motif probe R: AATTTTTATTTTAAA; Mutated probe F: CCCGGGGCGGGGGCC; Mutated probe R: GGCCCCCGCCCCGGG) were incubated in Binding buffer by rotating at 37 °C for 4 h. Samples were diluted with loading buffer and loaded onto BeyoGel™ EMSA PAGE (Beyotime, CAT#GS302S). Electrophoresis was performed at 100 V for 1 h, followed by transfer to nylon membrane (Beyotime, CAT#FFN13) at 60 V for 1 h with a Bio-Rad transfer unit (Bio-Rad). After transfer, DNA was immobilized with UV cross-linker. The membrane was blocked with blocking buffer for 15 min at room temperature and then incubated with Streptavidin-HRP (LI 925-32230, LI-COR® BIOSCIENCES, USA) for 15 min. Membrane was washed three times with washing buffer and imaged by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Han F., Yang B., Chen Y., Liu L., Cheng X., Huang J., Zhou K., Zhang D., Xu E., Lai M., Lv B., Cheng H, & Zhang H. (2023). Loss of GLTSCR1 causes congenital heart defects by regulating NPPA transcription. Angiogenesis, 26(2), 217-232.

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HEK293T Cell Lysis and Protein Degradation

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HEK293T cells were maintained in DMEM culture medium supplemented with 10% (v/v) FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified incubator with 5% CO₂ at 37 °C. Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail and PMSF. The cell lysis was centrifuged at 10,000 × g for ten minutes. One tenth the volume of the supernatants was pipetted as input samples. The remaining supernatants were incubated with M2 Magnetic Beads (M8823, Sigma) overnight by the standard protocol. For protein degradation assay, 10 μM MG132 was added to the culture medium just before plasmid transfection and then incubated for 16–24 hours.

Chen R.Z., Cheng X., Tan Y., Chang T.C., Lv H, & Jia Y. (2020). An ENU-induced mutation in Twist1 transactivation domain causes hindlimb polydactyly with complete penetrance and dominant-negatively impairs E2A-dependent transcription. Scientific Reports, 10, 2501.

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Phosphorylation of FAM122A by CHK1

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293T cells were transfected with pHAGE-FAM122A-FLAG-HA and cellular lysates were generated 2 days post transfection with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, and protease and phosphatase inhibitor cocktail sets (Millipore)). Negative control lysates were generated by mock transfecting 293T cells. FLAG-HA-FAM122A was immunoprecipitated from cellular lysates using M2 Magnetic Beads (Sigma-Millipore) at room temperature for 2 hours. Immunoprecipitated FLAG-HA-FAM122A protein was added to CHK1 kinase reactions with 100 ng purified CHK1 (Millipore) and 20 uM ATP-g-S (Abcam). CHK1 was omitted from denoted reactions as a negative control. Kinase Reactions were conducted as previously described (Kim et al., 2020 ). Briefly, reactions were incubated at 30°C for 30 min and thiophosphate species were alkylated with p-nitrobenzyl mesylate (Abcam) for 2 hours at room temperature. 6x SDS buffer was added to the reactions, samples were boiled, and FAM122A phosphorylation was assessed by SDS-PAGE followed by immunoblot.

Li F., Kozono D., Deraska P., Branigan T., Dunn C., Zheng X.F., Parmar K., Nguyen H., DeCaprio J., Shapiro G.I., Chowdhury D, & D’Andrea A.D. (2020). CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress. Molecular cell, 80(3), 410-422.e6.

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Smurf2 and HIPK2 Ubiquitination Interaction

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HEK293T cells were transfected with FLAG‐labelled Smurf2, Myc‐labelled HIPK2, HA‐labelled Ubiquitination using Lipofectamine 3,000, followed by lysing in lysis buffer. M2 magnetic beads (catalogue number: A4596, Sigma‐Aldrich) were adopted for immunoprecipitation at 4°C overnight. After washing for four times, the conjugated proteins were detected by western blotting with primary antibodies against Smurf2, HIPK2, Flag, Myc, and HA.
For another experiment, the HL‐1 cell lysates (500 μg) were immunoprecipitated with anti‐IgG (#3900, Cell Signaling Technology) or anti‐Smurf2 (#12024, Cell Signaling Technology) and incubated with protein G‐Agarose beads (Yeasen, Shanghai, China) at 4°C overnight. Subsequently, washing with 1 mL lysis buffer was performed for four times. Finally, the samples were subjected to western blotting. Three independent experiments were conducted.

Li Y., He Q., He C., Cai C., Chen Z, & Duan J. (2023). Activating transcription factor 4 drives the progression of diabetic cardiac fibrosis. ESC Heart Failure, 10(4), 2510-2523.

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Protein-Protein Interaction Assay with FLAG-Tagged Constructs

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Three hundred picomoles of FLAG-tagged proteins (eIF4G 84-294 or eRF3a) were incubated with 250 pmol PABPC1 constructs in a final volume of 400 µL binding buffer (25 mM HEPES at pH 7.8; 150 mM NaCl; 2 mM MgCl₂; 0.1% NP-40; 0.01% Triton X-100) in the presence of magnetic beads coupled to anti-FLAG antibodies (M2 magnetic beads; Sigma). After incubation for 2 h at 4°C, beads were washed twice with 500 µL wash buffer (25 mM HEPES at pH 7.8; 300 mM NaCl; 2 mM MgCl₂; 0.1% NP-40; 0.2% Triton X-100) and coprecipitated proteins were eluted with 1x SDS loading buffer. 10% of the protein mix was used as input control, all samples were separated on 12% SDS–polyacrylamide gels and stained with Coomassie Brilliant Blue.

Fatscher T., Boehm V., Weiche B, & Gehring N.H. (2014). The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay. RNA, 20(10), 1579-1592.

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Protein extraction and co-immunoprecipitation

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Whole embryos and HUVECs were lysed in 1x Laemmli Sample Buffer (BIO-RAD, 1610747) with 100 mM NaCl, 50 mM DTT, and 1 μg/ml phosphatase inhibitor cocktail 3 (SigmaAlrich, P0044) mixed with glass beads, and shaken in an Eppendorf shaker at 2,000 RPM at 85°C for 10 min. For co-immunoprecipitations HEK293 cells were washed in 1xPBS and lysed in buffer containing 20 mM Tris-HCl pH 7.5, 20 mM NaCl, 1x Thermo Halt protease inhibitors, 0.5% IGEPAL CA-630, 1 mM DTT, 1 mM EDTA, and 0.1 mM MG-132. Lysates were sonicated on ice using Fisher Scientific FB120 at 5 min/10 sec/10 sec, rotated with M2-Magnetic beads (Sigma-Alrich, M8823) for 3 hours and washed 3 times with 20 mM Tris-HCl pH 7.5, 20 mM NaCl, 0.1% IGEPAL CA-630, and 1 mM EDTA.

Tsitsikov E.N., Phan K.P., Liu Y., Tsytsykova A.V., Kinter M., Selland L., Garman L., Griffin C, & Dunn I.F. (2023). TRAF7 is an essential regulator of blood vessel integrity during mouse embryonic and neonatal development. iScience, 26(8), 107474.

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In vitro Hybrid-Protein Binding Affinity Assay

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To measure the hybrid-protein binding affinities, we performed the in vitro pull-down by using the radioactivity-labeled 60bp-long RNA-DNA hybrid described above. Flag-tagged proteins were purified from mammalian cells and immobilized to M2 magnetic beads (Sigma). In each reaction, 62.5 fmol hybrid was incubated with a specific protein at 1:10 molar ratio, and 1× binding buffer (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA pH 8.0, 1 mM DTT, 0.1% NP-40, 0.1 mg/ml BSA) was added into the reaction to bring the total volume up to 150 μl. Samples were held on a rotator for 3 h at room temperature. After the binding reaction, magnetic beads were washed 3 times with 1× binding buffer to remove the unassociated hybrids. The hybrids still associated with the beads were eluted by proteinase K digestion which was performed in 10 μl 1× binding buffer with 0.1% SDS for 30 min at 37 °C. Eluted samples were analyzed by non-denaturing 6% polyacrylamide gel in 0.5× TBE buffer at 4°C followed by autoradiography imaging. Human RNaseH1 catalytic inactive mutant (D210N) and GFP served as positive and negative controls, respectively. Uncropped gel images are in Supplementary Fig. 1.

Crossley M.P., Song C., Bocek M.J., Choi J.H., Kousorous J., Sathirachinda A., Lin C., Brickner J.R., Bai G., Lans H., Vermeulen W., Abu-Remaileh M, & Cimprich K.A. (2022). R-loop-derived cytoplasmic RNA-DNA hybrids activate an immune response. Nature, 613(7942), 187-194.

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