HEK293T cells expressing FLAG tagged RHEB -WT, −T38A, -Y35N, -D60I, and KRAS-G12V were immunoprecipitated using anti-FLAG M2 magnetic beads (Sigma). Briefly, the cells were lysed with lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.4% CHAPS, 1X Complete EDTA-free protease inhibitor cocktail (Roche), 1 mM Na3VO4), 150 mM NaCl, 25 mM MgCl2), and the supernatant was cleared of cellular debris using centrifugation (16,000×g for 10 min). Cleared supernatant was mixed with anti-FLAG M2 magnetic beads (Sigma) for affinity purification. The beads were collected, washed four times with lysis buffer. The remaining bound proteins were eluted three times with lysis buffer containing 62 μg/mL of 3X FLAG peptide. Eluted proteins were concentrated using Amicon Ultra 0.5-ml centrifugal filters NMWL 10 K (EMD Millipore, Billerica, MA).
Anti flag m2 magnetic bead
Manufactured by Merck Group
Sourced in United States, Germany, Macao, China, United Kingdom
Anti-FLAG M2 magnetic beads are a laboratory tool used to facilitate the purification and immobilization of FLAG-tagged proteins. These beads are coated with a monoclonal antibody that specifically binds to the FLAG epitope, allowing for the selective isolation of FLAG-tagged proteins from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (56 mentions)
3xflag peptide, by Merck Group (48 mentions)
Protease inhibitor cocktail, by Roche (32 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (20 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (19 mentions)
Protease inhibitor cocktail, by Merck Group (18 mentions)
Complete protease inhibitor, by Roche (15 mentions)
Protease inhibitor, by Roche (14 mentions)
Anti flag antibody, by Merck Group (11 mentions)
Anti flag, by Merck Group (10 mentions)
Anti flag m2, by Merck Group (9 mentions)
Complete protease inhibitor cocktail, by Roche (9 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (9 mentions)
Protease inhibitor, by Merck Group (8 mentions)
Mg132, by Merck Group (8 mentions)
Dynabead, by Thermo Fisher Scientific (8 mentions)
Opti mem, by Thermo Fisher Scientific (8 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (8 mentions)
Cell lysis buffer, by Beyotime (7 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
753 protocols using anti flag m2 magnetic bead
1
Affinity Purification of FLAG-Tagged RHEB Variants
2
Identification of Membrane-Localized Proteins
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Cells were collected from 10 ml of overnight LB cultures containing 50 μM Mn and were lysed by 1 ml of lysis buffer (Tris-HCl, 50 mM EDTA, 1 mg/ml lysozyme) incubated at 37°C for 30 minutes. The samples were sonicated for 5 minutes and incubated with 10 U/ml DNase at 37°C for 30 minutes. 1% Triton X-100 was added, and then samples were incubated on ice for 2 hours before adding anti-FLAG M2 magnetic beads (Sigma). Co-IP samples were prepared as described in the protocol of the anti-FLAG M2 magnetic beads (Millipore Sigma). Samples were end-over-end rotated with anti-FLAG M2 magnetic beads at 30°C for 3 hours and then placed on a magnetic stand. Supernatants were discarded and the samples were washed three times with PBS buffer at 30 °C for 10 min. Samples were eluted be either heating at 95°C for 10 minutes or treatment with glycine (pH 3) for 30 minutes at room temperature. Proteins in the samples were checked by immunoblot and were identified by LC-MS/MS (Cornell Proteomics and Metabolomics Facility). Only proteins with at least 2 peptides were considered as positive identifications. Table 1 summarizes all membrane-localized proteins from three experiments.
3
Immunoprecipitation of FLAG-tagged Protein
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WalR-FLAG was immunoprecipitated from 25 ml (Phos-tag experiment) or 600 ml (mass spectrometry experiment) cultures at OD600~0.4 using anti-FLAG M2 magnetic beads (Sigma M8823) according to the manufacturer’s instructions with the following modifications. Initial lysis to break down the cell wall was performed in 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 μg/ml DNaseI, 3 mM MgCl2, 1x Halt phosphatase inhibitor cocktail (Pierce), 1 mM PMSF, and 1 mg/ml lysozyme. After 20 min of lysis on ice with periodic vortexing, cell pellets were spun down and resuspended in lysis buffer without lysozyme and applied to 0.1 mm silica beads (BioSpec, 11079101z) in pre-chilled screw cap tubes. Cells were lysed using a FastPrep-24 5G instrument (MP Biomedicals) using 4 runs of 6.5 m/s for 40 seconds with samples placed on ice for 3 min between each run. Lysates were cleared by centrifugation at 19,000 x g for 20 min at 4°C. Elutions from the anti-FLAG M2 magnetic beads were performed competitively by addition of the 3x-FLAG peptide (Sigma F4799).
4
FLAG-tagged Protein Pulldown Protocol
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The protocol for the pulldown of FLAG-tagged proteins was modified and performed using anti-FLAG M2 magnetic beads (Sigma-Aldrich Inc., St. Louis, MO, USA) as previously described by M. J. Amorim et al. (24 (link)). Confluent 6-well dishes of MDCK cells were transfected with 500 ng of pHW2000-FLAG-GFP-NP. After 24 to 48 h, cells were infected with influenza A WSN/33 virus at an MOI equal to 10. At 6 h postinfection, 20 μM FA-6005 was added to the infected cells. DMSO was added as a negative control. All samples were collected and lysed at 8 h p.i. in 500 μl of a solution containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail (Roche Life Science, Switzerland) on ice for 30 min. Cell lysates were precleared with mouse IgG agarose, and the supernatant was bound to mouse anti-FLAG M2 magnetic beads on ice for 2 h. Beads were washed before and after sample binding with a wash buffer containing 50 mM NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 0.5% NP-40, and 10% glycerol (Sigma-Aldrich Inc., St. Louis, MO, USA). Bound proteins were eluted by being boiled in SDS-PAGE sample buffer. Western blotting was performed and imaged as described above.
5
Protein-Protein Interaction Assays
6
Identifying Membrane Proteins by Co-Immunoprecipitation
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Cells were collected from 10 ml of overnight LB cultures containing 50 μM Mn and were lysed by 1 ml of lysis buffer (TrisHCl, 50 mM EDTA, 1 mg/ml lysozyme) incubated at 37°C for 30 minutes. The samples were sonicated for 5 minutes and incubated with 10 U/ml DNase at 37°C for 30 minutes. 1% Triton X100 was added, and then samples were incubated on ice for 2 hours before adding anti-FLAG M2 magnetic beads (Sigma). Co-IP samples were prepared as described in the protocol of the anti-FLAG M2 magnetic beads (Millipore Sigma). Samples were end-over-end rotated with anti-FLAG M2 magnetic beads at 30°C for 3 hours and then placed on a magnetic stand.
Supernatants were discarded and the samples were washed three times with PBS buffer at 30 °C for 10 min. Samples were eluted be either heating at 95°C for 10 minutes or treatment with glycine (pH 3) for 30 minutes at room temperature. Proteins in the samples were checked by immunoblot and were identified by LC-MS/MS (Cornell Proteomics and Metabolomics Facility). Only proteins with at least 2 peptides were considered as positive identifications.Table 1 summarizes all membrane-localized proteins from three experiments.
Supernatants were discarded and the samples were washed three times with PBS buffer at 30 °C for 10 min. Samples were eluted be either heating at 95°C for 10 minutes or treatment with glycine (pH 3) for 30 minutes at room temperature. Proteins in the samples were checked by immunoblot and were identified by LC-MS/MS (Cornell Proteomics and Metabolomics Facility). Only proteins with at least 2 peptides were considered as positive identifications.
7
Immunoprecipitation of FLAG-tagged Proteins
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HEK293T cells were transfected with the indicated pCAGGS vectors and TransIT-LT1 (Mirus). At 48 h post-transfection, the cells were lyzed in lysis buffer [20 mM Tris-HCl (pH7.5), 100 mM NaCl, 0.5% Triton X-100, and protease inhibitor cocktail (Sigma)] on ice for 10 min. After centrifugation at 15,300 g for 10 min at 4°C, the supernatant was collected. Co-immunoprecipitation was performed by incubation with antibody (4°C, overnight) and subsequently with Dynabeads Protein G (Life Technologies) for 20 min, or by incubation with anti-FLAG M2 magnetic beads (Sigma) at 4°C overnight. Rabbit anti-FLAG antibody (F7425; Sigma), or mouse anti-FLAG M2 antibody (F1804; Sigma) were used for the immunoprecipitation. Dynabeads Protein G beads were suspended in 6x Laemmli buffer [375 mM Tris-HCl, 9% SDS, 50% glycerol, 0.03% bromophenol blue, 6% 2-mercaptoethanol] and heated at 98°C for 5 min. anti-FLAG M2 magnetic beads were incubated in buffer [20 mM Tris-HCl (pH7.5), 100 mM NaCl] containing 100 μg/mL FLAG peptide (Sigma) and the supernatant was suspended in Laemmli buffer and heated at 98°C for 5 min.
8
Affinity Purification of Tagged Proteins
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Tissue samples grown for ∼2 d at room temperature were harvested, pulverized, and suspended overnight in purification buffer (150 mM HEPES at pH 7.4, 250 mM KCl, 1mM EDTA, 1mM DTT, 10% glycerol, protease inhibitor) with anti-Flag M2 magnetic beads (Sigma). The beads were washed six times with purification buffer, and bound proteins were eluted with 200 µg/mL Flag tag peptide (Sigma). Eluted proteins were concentrated with TCA precipitation, resolved on SDS-PAGE, and recovered for mass spectrometry analysis. To verify the mass spectrometry results, Neurospora mats were cut into discs and transferred to flasks with minimal medium (1× Vogel's, 2% glucose). After growing for 24 h at room temperature, the tissues were briefly cross-linked with formaldehyde (1% final concentration) for 15 min, stopped with 125 mM glycine, washed with 1× PBS, and harvested. Protein extraction and quantification, Western blot analysis, and immunoprecipitation assays were performed as described previously (Cheng et al. 2001 (link)). Lysates (2 mg) were incubated overnight with anti-Flag M2 magnetic beads (Sigma) and washed six times with wash buffer (same as lysis buffer except with 500 mM NaCl), and the elute proteins were analyzed with Western blot.
9
Profiling MYC and FLAG-tagged Protein Interactomes
10
Immunoprecipitation of EGFP-NleH and Eps8 in HEK293T
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HEK293T cells were seeded into 10-cm tissue culture dishes and cotransfected with 3 μg of one of pEGFP, pEGFP-NleH1, pEGFP-NleH2, or their respective PxxDY motif derivatives and 3 μg of pFlag-Eps8 or pFlag-Eps8531–822. Cells were incubated for 16 h before being harvested for analysis. Cells were washed with cold phosphate-buffered saline (PBS) and lysed for 30 min with lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 1% Triton x-100,10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, with 1× EDTA-free Complete Protease Inhibitor Cocktail [Roche]). Cellular debris was pelleted by centrifugation, and lysates were subjected to immunoprecipitation with anti-FLAG M2 magnetic beads (Merck) according to the manufacturer’s instructions. Briefly, supernatants were incubated with equilibrated anti-FLAG M2 magnetic beads at 4 °C for 3 h. Beads were washed with lysis buffer, and bound proteins were eluted by adding 3× FLAG peptide (100 μg/mL in lysis buffer; Merck) and incubating with rotation for a further 30 min. Eluates were magnetically separated from beads, and 4× lithium dodecyl sulfate (LDS) sample buffer (Thermo Fisher Scientific) supplemented with dithiothreitol (DTT) (50 mM) was added. Samples were boiled for 10 min before being subjected to PAGE and immunoblotting as described below.
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