The SCF components such as human influenza hemagglutinin (HA)-Skp1, Cul1, Myc-Rbx1, and FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) were transfected in HEK293T cells by using polyethylenimine. After 48 h of transfection, the cells were harvested and resuspended in lysis buffer (LB) (25 mM Tris–HCl, pH 7.5, 225 mM KCl and 1% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). The lysates were incubated with agarose anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) for 6 h at 4°C with rocking. Beads were washed with LB and the SCF complexes eluted with FLAG elution buffer (300 µg/ml of peptide FLAG in 10 mM HEPES pH 7.9, 225 mM KCl, 1.5 mM MgCl2, and 0.1% NP-40) for 1 h at 4°C with rocking. The eluates were stored in 15% glycerol at −20°C until use. To evaluate the purification of SCF complexes, immunoblotting was performed and probed using anti-Fbxo7 (ABN1038, Merck Millipore, Watford, UK), anti-HA (Abcam, Cambridge, UK), anti-Gsk3β (Santa Cruz Biotechnologies, CA, USA), anti-Tomm20 (Abcam, Cambridge, UK), or anti-myc (Cell Signaling Technologies, MA, USA). The concentration of the complexes was determined against known concentrations of BSA by Coomassie blue staining of the gel. The densitometry of the bands was determined by ImageJ.
M2 anti flag agarose beads
Manufactured by Merck Group
Sourced in United States
M2 anti-FLAG agarose beads are a type of laboratory equipment used for the purification and detection of proteins tagged with the FLAG epitope. The beads are composed of agarose, a polysaccharide derived from seaweed, and are coated with the M2 monoclonal antibody, which specifically binds to the FLAG tag. This allows for the efficient capture and recovery of FLAG-tagged proteins from various samples, such as cell lysates or protein extracts.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (3 mentions)
Anti ha, by Abcam (2 mentions)
Anti tomm20, by Abcam (2 mentions)
Anti gsk3β, by Santa Cruz Biotechnology (2 mentions)
Anti myc, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Transit 293 reagent, by Mirus Bio (2 mentions)
Protein a agarose beads, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Amicon ultracentrifugal tubes, by Merck Group (1 mentions)
Anti v5 agarose beads, by Merck Group (1 mentions)
Gfp trap agarose beads, by Proteintech (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Foetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 hplc, by Thermo Fisher Scientific (1 mentions)
Easy spray nanosource, by Thermo Fisher Scientific (1 mentions)
17 protocols using m2 anti flag agarose beads
1
Purification and Analysis of SCF Complexes
2
Purification of SCF Ubiquitin Ligase Complexes
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The SCF components such as human influenza hemagglutinin (HA)-Skp1, Cul1, Myc-Rbx1, and FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) were transfected in HEK293T cells by using polyethylenimine. After 48 h of transfection, the cells were harvested and resuspended in lysis buffer (LB) (25 mM Tris–HCl, pH 7.5, 225 mM KCl and 1% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). The lysates were incubated with agarose anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) for 6 h at 4°C with rocking. Beads were washed with LB and the SCF complexes eluted with FLAG elution buffer (300 µg/ml of peptide FLAG in 10 mM HEPES pH 7.9, 225 mM KCl, 1.5 mM MgCl2, and 0.1% NP-40) for 1 h at 4°C with rocking. The eluates were stored in 15% glycerol at −20°C until use. To evaluate the purification of SCF complexes, immunoblotting was performed and probed using anti-Fbxo7 (ABN1038, Merck Millipore, Watford, UK), anti-HA (Abcam, Cambridge, UK), anti-Gsk3β (Santa Cruz Biotechnologies, CA, USA), anti-Tomm20 (Abcam, Cambridge, UK), or anti-myc (Cell Signaling Technologies, MA, USA). The concentration of the complexes was determined against known concentrations of BSA by Coomassie blue staining of the gel. The densitometry of the bands was determined by ImageJ.
3
Affinity Purification for Protein Identification
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A total of 4 × 108 cells were lysed in lysis containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2.5 mM EDTA, 0.5% NP-40, 0.2 mM PMSF, and 0.1 mM cocktail. Cell lysates were precleared with the protein A-agarose beads (sc-2003, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h and then incubated with the anti-Flag agarose M2 beads (F2426, Sigma-Aldrich, St. Louis, MO, USA) at 0.5 mL of beads per 100 mg of cell lysate for 2 h to overnight with rotation. The M2 beads were washed four times with buffer BC500 containing 20 mM Tris-HCl (pH 7.8), 500 mM KCl, 0.2 mM EDTA, 10% glycerol, 10 mM mercaptoethanol, 0.2% NP-40, 0.2 mM PMSF, and 0.1 mM cocktail. The protein complex was eluted with the Flag peptides (Sigma-Aldrich, St. Louis, MO, USA) at 0.2 mg/mL in buffer TBS containing 20 mM Tris-HCl (pH 7.8), 50 mM NaCl,0.2 mM PMSF, and 0.1 mM cocktail. The eluted proteins were resolved on 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for Western blotting and silver and colloidal staining analyses. The proteins located between 40-kDa and 55-kDa were excised from the gels and identified by standard mass spectrometry.
4
Analysis of NTHL1-XPG Interaction
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To analyze NTHL1 association with XPG, HBEC cells were grown and transiently transfected with NTHL1-Flag or control vector following manufacturer's protocol (Promega). Forty-eight hours after transfection, cells were subject to either mock or 10 mM H2O2 treatment for 10 min and were washed with PBS. Cells were incubated in complete DMEM medium for 10 min and were collected by trypsinization and lysates were prepared with a lysis buffer containing 50 mM HEPES–KOH (pH 7.5), 250 mM KCl, 0.5% Triton X-100, 2.5 mM MgCl2, 0.5 mM EDTA, 5% glycerol and protease inhibitor cocktail (Roche). The DNA and RNA in the suspensions was digested with 25 U/μl benzonase (Novagene) for 30 min on ice. The suspensions were cleared by centrifugation at 12 000 rpm for 30 min, and the supernatants were collected and used for immunoprecipitation assay with anti-FLAG agarose M2 beads (Sigma). After extensive washing of the M2 beads with the same buffer, NTHL1-Flag protein complexes were eluted in the wash buffer containing 0.4 μg/μl FLAG-peptide (Sigma). The samples were separated by 4–12% acrylamide gel (Novex) and analyzed by immunoblotting including detection with ECL (GE Healthcare) system.
5
Production and Purification of FLAG-WNT8a Protein
6
Immunoprecipitation and Western Blot Analysis
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Cells were lysed with RIPA lysis buffer supplemented with phosphatase inhibitors (1 mM Na3VO4, 2 mM NaF and 200 μM sodium pyrophosphate). Cell lysates containing equal amount of proteins were incubated with anti-Flag agarose beads (M2; Sigma-Aldrich), anti-V5 agarose beads (Sigma-Aldrich) or GFP-Trap agarose beads (Chromotek) at 4 °C for 2 h. Alternatively, cells were lysed with NP40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP40, 1% sodium deoxycholate, 1 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM PMSF) supplemented with phosphatase inhibitors. Cell lysates were pre-cleared with protein A Sepharose (GE Healthcare) at 4 °C for 1 h and incubated with various antibodies at 4 °C for overnight, followed by 2 h of incubation with protein A Sepharose at 4 °C. After washing the beads with lysis buffer for three times, proteins bound on beads were analyzed by western blot.
7
Protein Interaction Analysis by Co-Immunoprecipitation
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The HCC cells transfected with the abovementioned plasmids were harvested and lysed in cell lysate buffer (50 millimoles per liter (mM) Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; and 1% NP40) with complete protease inhibitors. Two milligrams of protein was incubated with anti-Flag agarose beads (M2, Sigma-Aldrich) at 4 °C overnight to pull down the corresponding exogenous proteins. For co-immunoprecipitation of the endogenous proteins, anti-ERK1, anti-ERK2, and anti-BCKDK antibodies were preincubated with protein A/G agarose (Pierce, Rockford, IL, USA) for 8 h. Normal rabbit IgG (CST) and Normal mouse IgG (Santa Cruz) were used as controls. Proteins were immunoprecipitated with the sepharose-antibody complexes on a rotator at 4 °C overnight. The beads were washed five times with cell lysate buffer and collected by centrifugation at 1000 grams (g), and the immunoprecipitates were subjected to Western blot with the corresponding antibodies. The primary antibodies used for immunoprecipitation are provided in Table S5 . Light chain-specific mouse anti-rabbit (#93702, CST) and rabbit anti-mouse (#58802, CST) IgG were used as secondary antibodies for immunoblotting.
8
Purification of SMARCAL1 Protein from HEK293T Cells
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Mammalian expression vector pcDNA3.1+/C-(K)-DYK containing human SMCARCAL1 cDNA (OHu16376) was purchased from Genscript (Piscataway, NJ). HEK293T cells were grown in the high glucose DMEM (Gibco) supplemented with 10% foetal bovine serum (Atlanta Biologicals), 1 mM sodium pyruvate, 1% penicillin and 1% streptomycin at 37 °C in the presence of 5% CO2. Cells were transiently transfected for 48 h using lipofectamine 2000 (Invitrogen). Cells were washed with phosphate-buffered saline (Gibco), then resuspended in the ice-cold lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol and 1 mM phenylmethanesulphonyl fluoride] and incubated at 4 °C for 30 min. After centrifugation, clarified cell lysate was mixed with M2 anti-FLAG agarose beads (Sigma-Aldrich) and then incubated at 4 °C for 2 h. Beads were then washed with lysis buffer, resuspended in the elution buffer [lysis buffer with 150 µg/ml of 3 × FLAG peptides (Sigma-Aldrich)] and incubated at 4 °C for 30 min. Eluted protein was immediately divided into small aliquots and preserved at −80 °C.
9
Immunoprecipitation and Mass Spectrometry Analysis
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The chromatin fractions were incubated with approximately 30 μL pre-equilibrated M2 anti-FLAG agarose beads (Sigma), rotating at 4°C overnight. After incubation, the flow-through was collected and the beads were washed with 40 CV IP wash buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 150 mM NaCl, 0.05% NP-40, 1x Protease inhibitor mix, PhosphoStop phosphatase inhibitor). Immunoprecipitates were eluted using 500 μg/ml 3x FLAG peptide (The Francis Crick Institute), dissolved in IP wash buffer, by incubation for 30 min at 4°C . 30 μL of elution fractions were subjected to SDS-PAGE and subsequently stained with Instant Blue Coomassie. The stained proteins were excised from the polyacrylamide gel, cut into 8 equal slices and submitted to mass spectrometry analysis. Proteins were in-gel digested with trypsin using a Janus Automated Workstation (Perkin Elmer) and peptides were analyzed using a LTQ Orbitrap-Velos mass spectrometer coupled to an Ultimate3000 HPLC and equipped with an EASY-Spray nanosource (ThermoFisher Scientific).
10
Purification of γ-Secretase Complexes
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