The nuclear fraction of cells transfected with WT-NP, NPbd3, or No NP were incubated with Anti- FLAG M2 Affinity Gel agarose beads (Sigma-Aldrich), incubated overnight at 4 °C with rotation, washed five times in RSB (reticulocyte standard buffer: 10 mM Tris HCl pH 7.5, 10 mM KCl, 1.5 mM MgCl2) + 0.2% NP-40, and eluted for 1 h at room temperature with 150 ng/ul 3X FLAG peptide (APEXBIO).
Anti flag m2 affinity gel agarose beads
Manufactured by Merck Group
The Anti-FLAG M2 affinity gel agarose beads are a chromatography resin used for the purification of FLAG-tagged proteins. The beads consist of agarose with covalently immobilized anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag fused to the target protein. This allows for the selective capture and purification of FLAG-tagged proteins from complex mixtures.
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5 protocols using anti flag m2 affinity gel agarose beads
1
Affinity Purification of Nuclear Proteins
2
Purification of Telomerase Enzyme
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Telomerase was purified via the 3xFLAG tag on hTERT encoded pVan107 using ANTI-FLAG M2 affinity gel agarose beads (Sigma Aldrich), as described previously with some modification (Fouquerel et al., 2016 (link)). An 80 μL bead slurry (per T75 flask) was washed three times with 10 volumes of 1X human telomerase buffer in 30% glycerol with 1 min centrifugation steps at 3500 r.p.m. at 4°C. The bead slurry was added to the lysate and nutated for 4–6 hr at 4°C. The beads were harvest by 1 min centrifugation at 3500 r.p.m, and washed 3X with 1X human telomerase buffer with 30% glycerol. Telomerase was eluted from the beads using 2x the bead volume of 250 μg/mL 3X FLAG peptide (Sigma Aldrich) in 1X telomerase buffer with 150 mM KCl. The bead slurry was nutated for 30 min at 4°C. The eluted telomerase was collected using Mini Bio-Spin Chromatography columns (Bio-Rad). Samples were flash frozen and stored a −80°C.
3
Immunopurification of Telomerase Complex
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Telomerase was immunopurified as previously described39 (link). Cells expressing hTR and 3xFLAG-tagged hTERT were harvested 48 hours after transfection and lysed in CHAPS lysis buffer (10 mM Tris-HCl, 1 mM MgCl2, 1 mM EDTA, 0.5% CHAPS, 10% glycerol, 5 mM β-mercaptoethanol, 120 U RNasin plus (Promega), 1 μg/ml each of pepstatin, aprotinin, leupeptin and chymostatin, and 1 mM AEBSF) for 30 min at 4°C. Sigma Anti-FLAG M2 affinity gel agarose beads (75 μl of a 1:1 slurry) were pre-washed with 1x human telomerase buffer (50 mM Tris-HCl, pH 8, 50 mM KCl, 1 mM MgCl2, 1 mM spermidine, 5 mM β-mercaptoethanol) with 30% glycerol and mixed with 500 μl of cell lysate for 3 hours at 4°C with rotation. Beads were harvested by centrifugation at 3,500 rpm for 1 min, washed three times with 1x telomerase buffer with 30% glycerol, flash frozen as a 1:1 slurry in 1x telomerase buffer with 30% glycerol, and stored at −80°C.
4
Immunoprecipitation of FLAG-tagged Proteins
5
Immunopurification of Telomerase Complex
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