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Anti flag m2 magnetic resin

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The Anti-FLAG M2 magnetic resin is a lab equipment product used for the purification and detection of proteins tagged with the FLAG epitope. It consists of an agarose-based resin with immobilized anti-FLAG M2 monoclonal antibodies, which have a high affinity for the FLAG tag. The resin can be used in various chromatographic techniques to capture and isolate FLAG-tagged proteins from complex samples.

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Lab products found in correlation

Flag peptide, by Merck Group (4 mentions) Chemidoc, by Bio-Rad (2 mentions) P53 antibody do 1, by Santa Cruz Biotechnology (1 mentions) Slide a lyzer, by Thermo Fisher Scientific (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor, by Merck Group (1 mentions) Protease inhibitor, by Roche (1 mentions) Anti flag m2 agarose resin, by Merck Group (1 mentions) Profinity imac ni charged resin, by Bio-Rad (1 mentions) Talon metal affinity resin, by Takara Bio (1 mentions) Anti gst, by Santa Cruz Biotechnology (1 mentions) Anti flag m2, by Merck Group (1 mentions) Myc trap resin, by Proteintech (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Gfp trap, by Proteintech (1 mentions) Edta free protease inhibitor cocktail, by Merck Group (1 mentions)

9 protocols using anti flag m2 magnetic resin

1

Affinity Purification of 53BP1 and p53 Complexes

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Cells lyzed in Benzonase Lysis Buffer (20 mM HEPES [pH 7.9], 40 mM KCl, 2 mM MgCl2, 12% glycerol, 0.5% CHAPS, 50 U/ml Benzonase [Novagen], 0.05% [v/v] phosphatase inhibitors [Sigma-Aldrich] and protease inhibitors [Roche]) were supplemented with KCL to a 450 mM final concentration and gently mixed for 30 min at 4°C. Clarified lysates were then cassette dialyzed (Slide-A-Lyzer, Thermo Fisher Scientific) in dialysis buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 10% Glycerol, 0.5 mM DTT, 0.5 mM PMSF, 5 mM NaF, 10 mM β-glycerolphosphate). Flag-HA-53BP1 or endogenous p53 complexes were purified from 1–2 mg total protein using anti-FLAG M2 magnetic resin (Sigma-Aldrich) or p53 DO-1 antibody (Santa Cruz Biotechnology) coupled to protein G Dynabeads (Invitrogen). Protein-bead complexes washed extensively in dialysis buffer were either boiled in Laemmli buffer or eluted in 3× Flag peptide (Sigma-Aldrich).
Cuella-Martin R., Oliveira C., Lockstone H.E., Snellenberg S., Grolmusova N, & Chapman J.R. (2016). 53BP1 Integrates DNA Repair and p53-Dependent Cell Fate Decisions via Distinct Mechanisms. Molecular Cell, 64(1), 51-64.
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2

RIG-I Protein Binding RNA Identification

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SH-SY5Y RIG-I complement cells were treated with siRNA for 48 h. Doxycycline was added to induce RIG-I expression for the final 24 h. Cells were washed twice in cold PBS, crosslinked with 0.5% formaldehyde for 10 min in PBS, quenched with 0.3M glycine for 5min, and washed twice with cold PBS. Cells were resuspended in RIPA buffer (50mM Tris [pH 8.0], 0.5% sodium deoxycholate, 0.05% SDS, 1mM EDTA, 150mM NaCl, 1mM DTT, 1% NP-40) and kept on ice for 20min followed by sonication. Anti-Flag M2 magnetic resin (Sigma) was washed in RIPA buffer followed by the addition of soluble extract and incubation at 4°C for 3 h. Beads were washed three times for 10 min at 4°C and then two times for 5 min at room temperature in RIPA buffer with 0.1%SDS, 1M NaCl, and 1M urea. Resin was eluted with 1X FLAG peptide (Sigma) in RIPA buffer for 45 min at 4°C. Crosslinks were reversed by adding 100 mM Tris [pH 8], 10mM EDTA, 1% SDS and 2% DTT to samples and heated at 70°C for 45 min. RNA was recovered by TRIzol extraction, isopropanol precipitation, DNase treatment, PCA extraction, and ethanol precipitation. Bound RNAs were analyzed by RT-qPCR.
Dunker W., Ye X., Zhao Y., Liu L., Richardson A, & Karijolich J. (2021). TDP-43 prevents endogenous RNAs from triggering a lethal RIG-I-dependent interferon response. Cell reports, 35(2), 108976.
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3

Affinity-based Protein Purification

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Anti-p-MEK1/2 Ser217/221 (#9121) and anti-HSP70 (#4873S) were purchased from Cell Signaling. Anti-p-BRAF T599 (#PA5–37497) was purchased from Invitrogen. Anti-FLAG M2 (#F1804), Anti-FLAG M2 agarose resin (#A2220), and Anti-FLAG M2 magnetic resin (#M8823) were purchased from Sigma Aldrich. Anti-GST (#SC-138) was purchased from Santa Cruz. Talon metal affinity resin (#635501) was purchased from Takara. Profinity IMAC Ni charged resin (#156–0131) was purchased from Bio-Rad.
Cope N., Candelora C., Wong K., Kumar S., Nan H., Grasso M., Novak B., Li Y., Marmorstein R, & Wang Z. (2018). Mechanism of BRAF Activation Through Biochemical Characterization of the Recombinant Full-Length Protein. Chembiochem : a European journal of chemical biology, 19(18), 1988-1997.
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4

RIG-I Protein Binding RNA Identification

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SH-SY5Y RIG-I complement cells were treated with siRNA for 48 h. Doxycycline was added to induce RIG-I expression for the final 24 h. Cells were washed twice in cold PBS, crosslinked with 0.5% formaldehyde for 10 min in PBS, quenched with 0.3M glycine for 5min, and washed twice with cold PBS. Cells were resuspended in RIPA buffer (50mM Tris [pH 8.0], 0.5% sodium deoxycholate, 0.05% SDS, 1mM EDTA, 150mM NaCl, 1mM DTT, 1% NP-40) and kept on ice for 20min followed by sonication. Anti-Flag M2 magnetic resin (Sigma) was washed in RIPA buffer followed by the addition of soluble extract and incubation at 4°C for 3 h. Beads were washed three times for 10 min at 4°C and then two times for 5 min at room temperature in RIPA buffer with 0.1%SDS, 1M NaCl, and 1M urea. Resin was eluted with 1X FLAG peptide (Sigma) in RIPA buffer for 45 min at 4°C. Crosslinks were reversed by adding 100 mM Tris [pH 8], 10mM EDTA, 1% SDS and 2% DTT to samples and heated at 70°C for 45 min. RNA was recovered by TRIzol extraction, isopropanol precipitation, DNase treatment, PCA extraction, and ethanol precipitation. Bound RNAs were analyzed by RT-qPCR.
Dunker W., Ye X., Zhao Y., Liu L., Richardson A, & Karijolich J. (2021). TDP-43 prevents endogenous RNAs from triggering a lethal RIG-I-dependent interferon response. Cell reports, 35(2), 108976.
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5

Affinity Purification of FLAG, GFP, and Myc Tagged Proteins

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FLAG immunoprecipitations were performed by harvesting cells and lysing in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40 and EDTA-free protease inhibitor cocktail (Sigma). Protein concentration in the resulting lysates was quantified by BCA assay (Thermo Scientific), and protein concentration was equalized before immunoprecipitation with anti-FLAG M2 magnetic resin (Sigma). One milliliter of whole-cell lysate, containing approximately 1.9 mg of protein, was incubated with 30 μl of anti-FLAG resin for 1 h at 4 °C. Samples were then washed three times in wash buffer [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40], then eluted by incubation with 250 μg/ml FLAG peptide (Sigma, cat. F3290). GFP and Myc immunoprecipitations were performed using GFP-Trap and Myc-Trap resin (ChromoTek), respectively, as in [61] (link). GST pull-down experiments were performed as described in [61] (link), using 40–100 pmol purified VPS33B/GST-VIPAR complex as bait (described above).
Hunter M.R., Hesketh G.G., Benedyk T.H., Gingras A.C, & Graham S.C. (2018). Proteomic and Biochemical Comparison of the Cellular Interaction Partners of Human VPS33A and VPS33B. Journal of Molecular Biology, 430(14), 2153-2163.
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6

Affinity Purification of FLAG-ALC1 and PARP1

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A 50 μL solution of 100 nM FLAG-ALC1, 50 nM unmodified or H3S10ADPr3 nucleosomes, and 100 nM PARP1 (unmodified, PARP1 SerADPrlong, or PARP1 SerADPrshort) in IP buffer (100 mM KCl, 25 mM HEPES pH 7.9, 2 mM MgCl2, 5% glycerol, 0.1% NP-40, 1 mM DTT) was incubated at 30 °C for 15 min. Binding reactions were then added to anti-FLAG M2 magnetic resin (MilliporeSigma; pre-equilibrated in IP Buffer) after keeping aside 5 μL as an input control for western blot analysis. Resin was incubated on an end-over-end rotator at 4 °C for 1 h, washed for three times for 1 min each with 0.5 mL of IP Buffer (with very gentle vortexing), and eluted via incubation in 2 X SDS loading dye at 95 °C for 5 min. Samples were run on 10% SDS PAGE Bis-Tris gels, analyzed via western blot and imaged on a BioRad ChemiDoc.
Mohapatra J., Tashiro K., Beckner R.L., Sierra J., Kilgore J.A., Williams N.S, & Liszczak G. (2021). Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling. eLife, 10, e71502.
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7

Affinity Purification of PARP1-FLAG Complexes

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A 100 μL solution of 5 μM FLAG-HPF1 (or HPF1D283A), 1 μM PARP1, 10 μM Olaparib, and 10 μM stimulating DNA in Pull-Down Buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 0.1% Triton X-100, 1 mM DTT) was incubated for 25 min at 25 °C. This solution was then centrifuged at 20,000 RCF for 10 min and the supernatant was added to 10 μL of Anti-FLAG M2 magnetic resin (MilliporeSigma; pre-equilibrated in Pull-Down Buffer), after keeping aside 30 μL from the reaction as an input control for SDS-PAGE gel analysis. Resin was incubated on an end-over-end rotator at 4 °C for 30 min, washed for 3 times for 1 min each with 0.5 mL of Pull-Down Buffer, and eluted via incubation in 2 X SDS loading dye at 95 °C for 5 min. Samples were analyzed on 10% SDS PAGE Bis-Tris gel and imaged via Coomassie Brilliant blue staining on a BioRad ChemiDoc.
Mohapatra J., Tashiro K., Beckner R.L., Sierra J., Kilgore J.A., Williams N.S, & Liszczak G. (2021). Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling. eLife, 10, e71502.
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8

Phosphorylation and Purification of Sld3/7 and Sld2

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Immediately prior to phosphorylation, FLAG-tagged Sld3/7 was diluted to 30 nM in 40 mM HEPES-KOH pH 7.6, 8 % glycerol, 400 μg/ml BSA, 0.02 % NP-40-S, 10 mM Mg-acetate, 2 mM DTT, 5 mM ATP with 310 mM K-glutamate (buffer PP + 310 mM K-glutamate). Sld2 was diluted to 120 nM in buffer PP + 235 mM K-glutamate. Budding yeast S-phase CDK (S-CDK) was added to 10 nM, and reactions incubated for 8 min at 25°C before addition of Sic1 to 220 nM. After 2 min incubation at 25°C, Sld2 mix was diluted 4x in buffer A + 0.5 M KCl. 5 μl magnetic anti-FLAG M2 resin (Sigma) washed with buffer A + 0.5 M KCl was added, and each sample incubated at 4°C for 30 min with rotation. Resin was washed 5x with 300 μl buffer A + 0.5 M KCl (Sld3/7) or +0.35 M KCl (Sld2), and resuspended in 10 μl of the same buffer supplemented with 0.25 mg/ml FLAG peptide. After shaking at 4°C for 30 min, supernatant was collected, aliquoted and frozen in liquid nitrogen for storage.
Douglas M.E., Ali F.A., Costa A, & Diffley J.F. (2018). The mechanism of eukaryotic CMG helicase activation. Nature, 555(7695), 265-268.
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9

Proteasome Reconstitution and Purification

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Proteasomes were reconstituted with 500 nM core particle and 1 μM tagless base, lid, and Rpn10, and allowed to assemble for 5 min in GF buffer with 1 mg/mL BSA, 5 mM ATP, and ATP regeneration system at room temperature. Magnetic ANTI-FLAG m2 resin (Sigma) was added to the solution and resin binding was allowed to proceed at 4°C for 1 hr. Resin was washed three times with 120 μL of GF buffer including 1 mg/mL BSA and 5 mM ATP, before eluting bound complexes with 35 μL of GF buffer supplemented with 5 mM ATP and 1 mg/mL 3X FLAG peptide at 30°C for 30 min.
Greene E.R., Goodall E.A., de la Peña A.H., Matyskiela M.E., Lander G.C, & Martin A. (2019). Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation. eLife, 8, e49806.
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