Anti flag m2 affinity agarose resin

Manufactured by Merck Group

The Anti-FLAG M2 Affinity Agarose resin is a laboratory product used for the purification and detection of FLAG-tagged proteins. It is composed of agarose beads that are cross-linked to the Anti-FLAG M2 antibody, which specifically binds to the FLAG peptide tag. The resin can be used for the affinity-based purification of FLAG-tagged proteins from a variety of sample sources.

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Lab products found in correlation

Protease inhibitor cocktail, by Roche (2 mentions) Pog44, by Thermo Fisher Scientific (1 mentions) Flp in t rex 293 cell, by Thermo Fisher Scientific (1 mentions) Ex cell 420 medium, by Merck Group (1 mentions) Bac to bacprotocols, by Thermo Fisher Scientific (1 mentions) S 200 superdex column, by GE Healthcare (1 mentions) Ni nta agarose resin, by Qiagen (1 mentions) Gfp trap beads, by Proteintech (1 mentions) Cb1001, by Merck Group (1 mentions) F3165, by Merck Group (1 mentions) Lambda protein phosphatase, by New England Biolabs (1 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (1 mentions) Bradford assay reagent, by Thermo Fisher Scientific (1 mentions) Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions) Azd8055, by Selleck Chemicals (1 mentions) Flag peptide, by Merck Group (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Complete edta free protease inhibitor cocktail, by Roche (1 mentions) Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) L glutamine, by Corning (1 mentions)

Close spelling variants of this product

Anti-FLAG M2 affinity resin Anti-FLAG M2 affinity agarose Anti-FLAG agarose affinity resin FLAG M2 affinity resin Anti-Flag affinity resin FLAG M2 resin M2 agarose resin Flag M2 agarose Anti-FLAG M2 Anti-FLAG resin

8 protocols using anti flag m2 affinity agarose resin

Affinity Purification of FLAG-Tagged Proteins

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Anti-FLAG M2 Affinity Agarose resin (Sigma, ∼30 μl) was washed 3× with 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl and then incubated with clarified cell lysate overnight at 4°C with gentle agitation. Agarose beads were collected by centrifugation at 1000×g for 1 min and then washed 5× with 50 mM Tris–HCl (pH 8.0) containing 150 mM NaCl. Precipitated (bead-bound) proteins were eluted by incubation with 2× SDS sample loading buffer for 5 min at 98°C.

Byrne D.P., Clarke C.J., Brownridge P.J., Kalyuzhnyy A., Perkins S., Campbell A., Mason D., Jones A.R., Eyers P.A, & Eyers C.E. (2020). Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signalling networks using SILAC-based phosphoproteomics. Biochemical Journal, 477(13), 2451-2475.

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Mapping Protein-Protein Interactions in Reprogramming

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For co-IP in MEFs, MEFs were infected with indicated lentivirus expressing individual reprogramming factors, followed by induction by doxycycline at 1μg/ml for 48 hours. For co-IP in 293 cells, FLAG-HA-tagged mouse O, S, K and M cDNAs were cloned into pcDNA5/FRT/TO (Invitrogen). Cell lines stably expressing these factors were generated by co-transfection of these individual constructs with pOG44 (Invitrogen) into Flp-In T-REx-293 cells (Invitrogen), followed by hygromycin B selection. Selected clones were induced by doxycycline at 1μg/ml for 48 hours before Co-IP. To map Sox2 binding, indicated truncation mutants were generated in pcDNA5/FRT/TO with FLAG-HA tag, and transiently transfected into 293T cells for co-IP. Cells were harvested and lysed in a buffer containing BC300 (50 mM Tris [pH 7.4], 300 mM KCl, 20% glycerol, 0.2mM EDTA), 0.1% NP40 and 1x protease inhibitor cocktail (Roche). Cleared lysates were diluted by equal volume of BC0 (50 mM Tris [pH 7.4], 20% glycerol, 0.2mM EDTA) and protease inhibitors, so that the final condition was identical to the IP buffer (BC150, 0.05% NP40 and protease inhibitors). Co-IP was carried out by rocking the diluted lysates with anti-FLAG M2 affinity agarose resin (Sigma) at 4°C overnight, followed by thorough wash in the IP buffer. Washed resin was boiled with 1x SDS gel loading buffer and used in western blotting.

Yang Z., Augustin J., Hu J, & Jiang H. (2015). Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors. PLoS ONE, 10(12), e0145336.

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Recombinant Nectin-4 Protein Expression

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HT1376, NCI-H292, PC3, and
CT26 cells were obtained from ATCC (American Type Culture Collection).
MC38 cells were obtained from the National Cancer Institute (L-159-2018/1).
CT26 and MC38 cells were engineered to express mouse Nectin-4 (NM_027893.3)
as described.^{21 (link)} Human peripheral blood
mononuclear cell (PBMC) isolation was described.^{21 (link)}Recombinant proteins: human CD137 (92 204B, R&D
Systems) and human CD137L (2295-4L-025, R&D Systems) were purchased.
Human Nectin-4 (residues 32–349) and rat Nectin-4 (residue
31–347) with a gp67 signal sequence and C-terminal FLAG tag
were cloned into pFastbac-1 and baculovirus using standard Bac-to-Bac
protocols (Life Technologies). Sf21 cells at 1 × 10⁶ mL^–1 in Excell-420 medium (Sigma) at 27 °C
were infected at a multiplicity of infection (MOI) of 2 with a P1
virus stock for protein expression. The supernatant was harvested
at 72 h and incubated for 1 h at 4 °C with anti-FLAG M2 affinity
agarose resin (Sigma) followed by a phosphate-buffered saline (PBS)
wash. Resin was subsequently transferred to a column and washed extensively
with phosphate-buffered saline (PBS). Protein was eluted with 100
μg/mL FLAG peptide concentrated to a volume of 2 mL and loaded
onto an S-200 Superdex column (GE Healthcare) in PBS at 1 mL/min.
2 mL fractions were collected and fractions containing Nectin-4 protein
were concentrated.

Upadhyaya P., Kristensson J., Lahdenranta J., Repash E., Ma J., Kublin J., Mudd G.E., Luus L., Jeffrey P., Hurov K., McDonnell K, & Keen N. (2022). Discovery and Optimization of a Synthetic Class of Nectin-4-Targeted CD137 Agonists for Immuno-oncology. Journal of Medicinal Chemistry, 65(14), 9858-9872.

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Enrichment and Purification of RhopH Complex

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Up to 1 mL of enriched schizont-stage parasites were harvested by the percoll–sorbitol method and frozen in liquid nitrogen at 20% v/v in 200 mM NaCl, 10 mM Tris, pH 7.5 with 1 mM phenylmethylsulfonyl fluoride (PMSF). Frozen parasites were thawed at room temperature, and insoluble debris was pelleted at 20,000 × g for 10 min at 4°C. NaCl was added to 500 mM before overnight incubation of the clarified lysate with anti-FLAG M2 affinity agarose resin (Sigma–Aldrich) at 4°C with gentle agitation. The resin was subsequently washed with 1–5 mL of 10 mM Tris, pH 7.5 and 500 mM NaCl before elution in 10 mM Tris, pH 7.5, 200 mM NaCl and 0.15 mg/mL 3xFLAG peptide. The eluate was concentrated for native mass spectrometry and cryo-EM studies via a second affinity purification on Ni-NTA agarose resin (Qiagen) and small volume elution in 200 mM NaCl, 300 mM imidazole, 10 mM Tris, pH 7.5. After overnight dialysis to remove imidazole, purified RhopH complex was further concentrated by ultracentrifugation at 150,000 × g for 1 hr, yielding 0.8–2 mg/mL protein in 30 µL.

Schureck M.A., Darling J.E., Merk A., Shao J., Daggupati G., Srinivasan P., Olinares P.D., Rout M.P., Chait B.T., Wollenberg K., Subramaniam S, & Desai S.A. (2021). Malaria parasites use a soluble RhopH complex for erythrocyte invasion and an integral form for nutrient uptake. eLife, 10, e65282.

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Affinity Purification of Protein Complexes

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Cell lysates were adjusted to 1 μg/μl in lysis buffer. Either GFP-trap beads (ChromoTek) or Anti-FLAG-M2-Affinity agarose resin (SigmaAldrich) was equilibrated with lysis buffer. 300-500 μg of total protein was added to 10-15 μl of beads (50% slurry) and incubated for an hour at 4°C under agitation. Centrifugation steps at 200xg were done at 4°C for 2 minutes. Supernatant (flowthrough) was separated from beads, and beads were washed 3-5 times in lysis buffer. Proteins were eluted in lysis buffer containing Laemmli SDS sample buffer by boiling at 95°C for 5 minutes.

Röth S., Macartney T.J., Konopacka A., Chan K.H., Zhou H., Queisser M.A, & Sapkota G.P. (2020). Targeting Endogenous K-RAS for Degradation through the Affinity-Directed Protein Missile System. Cell Chemical Biology, 27(9), 1151-1163.e6.

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Affinity Purification of FLAG-Tagged Proteins

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Anti-FLAG M2 Affinity Agarose resin (Sigma, ~30 μL) was washed 3x with 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl and then incubated with clarified cell lysate overnight at 4 °C with gentle agitation. Agarose beads were collected by centrifugation at 1,000 x g for 1 min and then washed 5x with 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. Precipitated (beadbound) proteins were eluted by incubation with 2x SDS sample loading buffer for 5 min at 98 °C.

Byrne D.P., Clarke C.J., Brownridge P., Kalyuzhnyy A., Perkins S., Campbell A., Mason D.Y., Jones A.R., Eyers P.A., & Eyers C.E. (2020). Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics.

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Affinity Purification of Protein Complexes

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Individual wells of a 6 well-dish were transfected with pre-mixed pFlag-GFP, pCCF-v-src, pCCF-E. americana -HopBF1 D170A and pcDNA-V5-HSP90β or the S108E mutant plasmids using PolyJet transfection reagent per manufacturer’s instructions. Fresh media was added 5 hours after transfection. Cells were harvested the following day and lysed in ice-cold HENG buffer supplemented with a protease inhibitor cocktail (Roche). Whole cell lysates were then normalized by the total amount of protein. Anti-Flag M2 agarose affinity resin (Sigma) was added to the lysates and incubated for 3 hours at 4°C on an orbital shaker. The resin was pelleted by centrifugation and washed four times with ice-cold HENG buffer. Immunoprecipitated proteins were then eluted using 3X Flag peptide diluted in HENG buffer, filtered to remove contaminating resin (Millipore, Durapore-PVDF 22 μm; UFC30GV00) then concentrated (Millipore, Amicon Ultra-0.5mL 10Kda UFC501096). SDS loading buffer containing 1% β-mercaptoethanol was added to the samples and boiled. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with anti-Flag M2 (F3165, Sigma), anti-V5 (R960, Thermo Fisher Scientific), and anti-GAPDH (CB1001, EMD Millipore) antibodies. Results shown are representative of at least three independent experiments.

Lopez V.A., Park B.C., Nowak D., Sreelatha A., Zembek P., Fernandez J., Servage K.A., Gradowski M., Hennig J., Tomchick D.R., Pawłowski K., Krzymowska M, & Tagliabracci V.S. (2019). A bacterial effector mimics a host HSP90 client to undermine immunity. Cell, 179(1), 205-218.e21.

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Protein Complex Characterization Protocol

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Antibodies: anti-Rictor (2114, CST), anti-mTOR (2972, CST), anti-HA (3724, CST), anti-Akt (4691, CST), anti-phospho-Ser473 Akt (4060, CST), anti-phospho-Ser422 SGK1 (SC-16745-R, SCBT), and horseradish peroxidase–labeled anti-rabbit secondary antibodies (7074, CST).
Other reagents: anti-FLAG M2 agarose affinity resin (A2220, Sigma), 3×FLAG peptide (F4799, Sigma), Expi29 expression medium (A1435101, Thermo Fisher Scientific), ExpiFectamine 293 transfection kit (A14525, Thermo Fisher Scientific), Opti-MEM I reduced serum medium (31985062, Thermo Fisher Scientific), dulbecco’s modified eagle medium (DMEM) with high glucose (4.5 g/Liter) (CCFAA005, UCSF Cell Culture Facility), fetal bovine serum (11650, Atlanta Biologicals), penicillin-streptomycin (30-002-CI, Corning), L-glutamine (25-005-CI, Corning), trypsin (25-052-CI, Corning), PEI (24765, Polysciences), complete EDTA-free protease inhibitor cocktail (11697498001, Roche), PhosSTOP phosphatase inhibitor (04906837001, Roche), Bradford assay reagent (1856209, Thermo Fisher Scientific), ECL Western blotting detection reagent (RPN2106, GE Heathcare), Lambda protein phosphatase (P0753, NEB), AZD8055 (S1555, Selleckchem), Superose 6 increase 3.2/300 Gl (29091598, GE Healthcare), and Superdex 200 increase 3.2/300 Gl (28990946, GE Healthcare).

Yu Z., Chen J., Takagi E., Wang F., Saha B., Liu X., Joubert L.M., Gleason C.E., Jin M., Li C., Nowotny C., Agard D., Cheng Y, & Pearce D. (2022). Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt. The Journal of Biological Chemistry, 298(9), 102288.

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