Anti flag m2 magnetic bead slurry

Manufactured by Merck Group

The Anti-FLAG M2 magnetic bead slurry is a laboratory product designed for the purification and detection of proteins tagged with the FLAG epitope. The beads are coated with the Anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag, allowing for the effective capture and isolation of FLAG-tagged proteins from complex samples.

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Lab products found in correlation

Anti flag antibody, by Merck Group (2 mentions) Protease inhibitor tablet, by Roche (2 mentions) Dykddddk, by Sino Biological (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Nanodrop instrument, by Thermo Fisher Scientific (2 mentions) Ripa buffer, by Merck Group (2 mentions) F7425, by Merck Group (1 mentions)

5 protocols using anti flag m2 magnetic bead slurry

FLAG-tagged Protein Immunoprecipitation

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Cells were lysed with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor tablet (Roche) and rotated for 30 minutes at 4°C. The cell lysate was then spun for 10 minutes at 15,000 rpm, 4°C. For cell fractionations, we performed REAP fractionation as described previously [38 (link)]. For each immunoprecipitation, 2.5 mg of protein was mixed with 20 μL of anti-FLAG M2 magnetic bead slurry (Sigma) in a 1 mL volume, using lysis buffer without detergent to fill the volume. The mixture was rotated overnight at 4°C and subsequently washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40) supplemented with protease inhibitor tablet (Roche), with 5 minutes of rotation at 4°C per wash. The beads were resuspended in 25 μL 2X Laemmli buffer and boiled to elute immunoprecipitants (IP). For western blot analysis, 1% input and 100% of IP were used for SDS-PAGE and probed with anti-FLAG antibody (1:3000, Sigma F7425).

Ly M., Burgess H.M., Shah S.B., Mohr I, & Glaunsinger B.A. (2022). Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing. PLoS Pathogens, 18(2), e1010099.

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Immunoprecipitation of AGO1-4 and Pan-AGO in ESCs and C2C12

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All immunoprecipitation in AGO1–4^−/− -FHAGO2 ESCs were carried out with 60 μl anti-FLAG M2 magnetic bead slurry (Sigma Aldrich) per 10×10⁶ cells, and scaled up accordingly, after cellular fractionation described above. Lysates were incubated with anti-FLAG M2 magnetic beads for 2 hr at 4°C. The immunoprecipitated material was washed three times with 1 mL NP-40 lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% (v/v) NP40) and collected each time on a magnetic rack. After the final wash, beads were resuspended in 60 μl 2x sample buffer and boiled at 95°C for 5 min. The supernatant was collected, and the beads discarded.
All pulldowns in C2C12 were carried out with 500 μg Pan-AGO affinity peptide or mutant affinity peptide and 60 μl anti-FLAG M2 magnetic bead slurry per 10×10⁶ cells, and scaled up accordingly (Hauptmann et al., 2015 (link)). Lysates were incubated with Pan-AGO affinity peptide for 1.5 hr and subsequently with anti-FLAG M2 magnetic beads for a further 1.5 hr at 4°C. The immunoprecipitated material was washed three times with 1 mL NP-40 lysis buffer and collected each time on a magnetic rack. After the final wash, beads were resuspended in 60 μl 2x sample buffer and boiled at 95°C for 5 min. The supernatant was collected, and the beads discarded.

Sarshad A.A., Juan A.H., Muler A.I., Anastasakis D.G., Wang X., Genzor P., Feng X., Tsai P.F., Sun H.W., Haase A.D., Sartorelli V, & Hafner M. (2018). Argonaute-miRNA Complexes Silence Target mRNAs in the Nucleus of Mammalian Stem Cells. Molecular cell, 71(6), 1040-1050.e8.

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Immunoprecipitation of FLAG-tagged Proteins

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Cells were lysed with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor tablet (Roche) and rotated for 30 minutes at 4°C. The cell lysate was then spun for 10 minutes at 15,000 rpm, 4°C. For each immunoprecipitation, 2.5 mg of protein was mixed with 20 μL of anti-FLAG M2 magnetic bead slurry (Sigma) in a 1 mL volume, using lysis buffer without detergent to fill the volume. The mixture was rotated overnight at 4°C and subsequently washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40) supplemented with protease inhibitor tablet (Roche), with 5 minutes of rotation at 4°C per wash. The beads were resuspended in 25 μL 2X Laemmli buffer and boiled to elute immunoprecipitants (IP). For western blot analysis, 1% input and 100% of IP were used for SDS-PAGE and probed with anti-FLAG antibody (1:3000, Sigma F7425).

Ly M., Burgess H.M., Mohr I., & Glaunsinger B.A. (2021). Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing.

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MAGI1 Immunoprecipitation from HEK293T Cells

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Plasmids were transiently transfected to HEK293T cells for transgene expression using FuGENE (4 µL FuGENE per 1 µg of DNA) in 100 µL of Opti-MEM (Gibco) per well of a six-well plate (2 µg DNA total per well). Media was replaced 24 hr later. Following an additional 24 hr incubation, cells were washed with PBS and collected by the addition of 250 µL of RIPA buffer (EMD Millipore) or non-denaturing lysis buffer (20 mM Tris, 137 mM NaCl, and 0.5% NP-40) with scraping. For MAGI1 immunoprecipitation, lysates were tip sonicated. Insoluble material was separated by centrifugation (18,253 × g, 5 min, 4 °C) and the protein concentration in cellular lysates was determined by absorbance on a nanodrop instrument (Thermo Fisher). 1 mg of lysate in 1 mL of lysis buffer was incubated overnight at 4 °C with 20 µL of anti-FLAG M2 magnetic bead slurry (MilliporeSigma, no. M8823). After three washes with lysis buffer, immunoprecipitated material was eluted with 250 µg per mL of FLAG peptide (DYKDDDDK, Sino Biological (Beijing, China)). Eluted material from immunoprecipitations and whole-cell lysates were evaluated by immunoblotting analyses as described above.

Bulos M.L., Grzelak E.M., Li-Ma C., Chen E., Hull M., Johnson K.A, & Bollong M.J. (2023). Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2. eLife, 12, RP88508.

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Plasmid Transfection and Immunoprecipitation

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Plasmids were transiently transfected to HEK293T cells for transgene expression using FuGENE (4 μL FuGENE per 1 μg of DNA) in 100 μL of Opti-MEM (Gibco) per well of a six-well plate (2 μg DNA total per well). Media was replaced 24h later. Following an additional 24-hour incubation, cells were washed with PBS and collected by the addition of 250 μL of RIPA buffer (EMD Millipore) or nondenaturing lysis buffer (20 mM Tris, 137 mM NaCl, and 0.5% NP-40) with scraping. For MAGI1 immunoprecipitation, lysates were tip sonicated. Insoluble material was separated by centrifugation (18,253 xg, 5 min, 4°C) and the protein concentration in cellular lysates was determined by absorbance on a nanodrop instrument (ThermoFisher). 1 mg of lysate in 1 mL of lysis buffer was incubated overnight at 4°C with 20 μL of anti-FLAG M2 magnetic bead slurry (MilliporeSigma, no. M8823). After three washes with lysis buffer, immunoprecipitated material was eluted with 250 μg per mL of FLAG peptide (DYKDDDDK, Sino Biological). Eluted material from immunoprecipitations and whole-cell lysates were evaluated by immunoblotting analyses as described above.

Bulos M.L., Grzelak E.M., Li-Ma C., Chen E., Hull M., Johnson K.A, & Bollong M.J. (2023). Pharmacological inhibition of CLK2 activates YAP by promoting alternative splicing of AMOTL2. bioRxiv.

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