To purify FLAG-tagged Polη (Polη-FLAG), 4 liters of yeast cells cultured in YEPR were grown to OD600 1.8. Benomyl (final 80 μg/ml) was added and when cells reached G2/M, two liters of cells were harvested as the “minus break” sample. Galactose (final 2%) was added to the remainder to induce DSBs by PGAL-HO on chromosome III, and chromosome VI with an ectopic integrated HO site. The remaining cells were collected as the “plus break” sample after 90 min. Cell pellets were then frozen with liquid nitrogen and stored at −80° until preparation of whole-cell extracts. Thawed pellets were resuspended in lysis buffer (25 mM HEPES-NaOH pH 7.9, 400 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, 1 mM PMSF, 10 mM sodium butyrate, protease inhibitor cocktail, and phosphatase inhibitor cocktail) and lysed in a 6870 freezer mill (SPEX, CertiPrep). Whole-cell extracts were cleared by centrifugation at 17,000 g for 30 min. The soluble fraction was incubated with equilibrated M2-FLAG affinity resin (Sigma) at 4° and rotated overnight to immunoprecipitate Polη-FLAG. The M2-FLAG affinity resin was then washed once with lysis buffer and Polη-FLAG was eluted through 3XFLAG peptide (Sigma) competition. Eluates were precipitated with TCA, resuspended in 8 M urea, and run on SDS-PAGE, followed by Coomassie staining before gel purification and nLC-MS/MS analysis.
Flag m2 affinity resin
Manufactured by Merck Group
The FLAG M2 affinity resin is a laboratory product designed for the purification of proteins that have been engineered to contain a specific FLAG tag sequence. The resin is composed of an agarose matrix with covalently coupled anti-FLAG monoclonal antibodies, which enable the selective capture and isolation of FLAG-tagged proteins from complex mixtures. This product is intended for use in various protein research and development applications requiring the purification of tagged proteins.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Direct zol miniprep kit, by Zymo Research (1 mentions)
Tri reagent, by Zymo Research (1 mentions)
Anti pan akt, by Cell Signaling Technology (1 mentions)
E3012, by Thomas Scientific (1 mentions)
Anti ck2α, by Cell Signaling Technology (1 mentions)
Anti ps473 akt, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Purexpress in vitro protein synthesis kit, by New England Biolabs (1 mentions)
Hsp60, by Abcam (1 mentions)
Oxphos cocktail, by Abcam (1 mentions)
Gfp trap, by Proteintech (1 mentions)
His6 ubiquitin, by R&D Systems (1 mentions)
Actin hrp, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Tla 120, by Beckman Coulter (1 mentions)
Benzonase, by Merck Group (1 mentions)
18 protocols using flag m2 affinity resin
1
Purification of FLAG-tagged Polymerase Eta
2
Ribosome Ubiquitination During ER Stress
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To assess ribosome ubiquitination during ER stress, ribosomes were purified with Myc-tagged ubiquitin (Myc-Ubi) and the FLAG-tagged ribosomal protein uL23 (uL23-FLAG), as described previously22 (link). Yeast cells harbouring pCUP1p-MYC-UBI and pRPL25(uL23)-FLAG were cultured in 800 mL of synthetic complete medium. To induce the expression of Myc-Ubi, the cells were cultured in the presence of 0.1 mM Cu2+ for 1 h, followed by the addition of Tm to a concentration of 1 µg/mL and harvesting at the indicated time points. Cell lysates were prepared, and FLAG-tagged ribosomes were purified using M2 FLAG-affinity resin (Sigma), as described previously22 (link). Affinity purified samples were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or western blotting with an anti-Myc antibody.
3
Purification of Hel2-FLAG Yeast Protein
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A yeast strain (see Supplementary Table 2 ) overexpressing Hel2-FLAG was grown in synthetic dropout media. Cells were harvested by centrifugation and subsequently disrupted by cryogenic milling (SPEX SamplePrep 6970EFM Freezer/Mill). Cell-powder was resuspended in lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 10 mM Mg(OAc)2, 0.01% NP-40, 1 mM PMSF/DTT, 1 protease inhibitor pill/50 ml (Roche, #04693132001)) and centrifuged at 30,596 x g for 30 min. The supernatant was purified using M2 FLAG affinity resin (Sigma-Aldrich, #A2220). During the washing steps, the salt concentration was decreased from 500 mM NaCl to 100 mM NaCl in 100 mM steps. Hel2-FLAG was eluted by incubation of the resin with 3x FLAG peptide in elution buffer (50 mM HEPES, 100 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, 0.05% Nikkol).
4
Purification of Gcn2 and eIF2α-ΔC from Yeast and E. coli
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Transformants of H2684 bearing plasmids pSL101, pSL102, pSL103, pSL104, pSL105, or pSL106 were grown to saturation in SC-Ura medium, diluted to A600 = 0.2 in SC-Ura containing 10% galactose as carbon source and grown to A600 ∼2.5. Cells were harvested (∼25 g), washed with cold distilled water containing EDTA-free protease inhibitor cocktail (PIC) (Boehringer Mannheim) and 0.5 mM PMSF, resuspended in ice-cold binding buffer (BB) (100 mM sodium phosphate [pH 7.4], 500 mM NaCl, 0.1% Triton X-100, EDTA-free PIC, 1 µg/ml leupeptin, and 1 mM PMSF) and disrupted using SPEX freezer mill (model 6870). Lysates were clarified by centrifugation at 39,000×g for 2 h at 4°C and mixed with 1 ml of M2-FLAG affinity resin (Sigma) overnight at 4°C. The resin was washed three times with 10 vol of BB and Gcn2 was eluted with 100 units of AcTEV protease in 500 µl of 1× TEV buffer (50 mM Tris pH 8, 0.5 mM EDTA, 1 mM DTT). The eluates were concentrated with an Amicon Centricon filter (exclusion limit of Mr 10,000) and dialyzed against 10 mM Tris-HCl [pH 7.4], 50 mM NaCl, 20% glycerol and stored at −80°C. The eIF2α−ΔC protein was purified from E. coli as previously described [13] (link).
5
RQT-mRNA Interaction Analysis
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For analysis of the RQT-mRNA interaction, 4-thiouridine (4SU)-containing mRNA was generated by addition of 7.5 mM 4SU to the in vitro transcription reaction. 8 pmol of ubiquitinated RNCs (stalled on CGN12 or CGN12-4SU mRNA) were incubated with a 10x molar excess of purified RQT complex at room temperature for 5 min. After crosslinking with UVA, samples were affinity-purified using M2 FLAG affinity resin (Sigma-Aldrich, #A2220). Binding of Slh1-ribosome complexes to the beads was performed in binding buffer (50 mM HEPES pH 7.5, 200 mM KOAc, 100 mM arginine, 125 mM sucrose, 1 mM PMSF) at 4 °C for 1 h. After two washing steps with binding buffer, again two washing steps with either 4 mM ATP or 5 mM EDTA were performed. Samples were eluted by addition 3xFLAG peptide in elution buffer (50 mM HEPES, 100 mM KOAc, 62.5 mm sucrose, 1 mM DTT). To remove remaining protein, samples were treated with proteinase K in ProtK buffer (100 mm Tris-HCl pH 7.5, 50 mm NaCl, 10 mM EDTA pH 8) at 37 °C for 20 min at a concentration of 1 mg/ml. Subsequently, equal volume of ProtK buffer containing 7 M urea was added for further incubation at 37 °C for 20 min. After addition of TRI reagent (Zymo Research #R2051), RNA was extracted using a Direct-zol Mini Prep kit (Zymo Research #R2051).
6
Purification of Gcn2 and eIF2α-ΔC proteins
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Transformants of H2684 bearing plasmids pSL101, pSL102, pSL526, pSL527, pSL529, pSL530 and pSL542 were grown to saturation in SC-Ura medium, diluted to A600 = 0.2 in SC-Ura containing 10% galactose as carbon source and grown to A600 of ∼2.5. Cells were harvested (∼25 g), washed with cold distilled water containing EDTA-free protease inhibitor cocktail (PIC) (Boehringer Mannheim) and 0.5 mM PMSF, resuspended in ice-cold binding buffer (BB) (100 mM sodium phosphate [pH 7.4], 500 mM NaCl, 0.1% Triton X-100, EDTA-free PIC, 1 μg/ml leupeptin, and 1 mM PMSF) and disrupted using SPEX freezer mill (model 6870). Lysates were clarified by centrifugation at 39,000 × g for 2 h at 4°C and mixed with 1 ml of M2-FLAG affinity resin (Sigma) overnight at 4°C. The resin was washed three times with 10 vol of BB and Gcn2 was eluted with 100 units of AcTEV protease in 500 μl of 1X TEV buffer (50mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1mM DTT). The eluates were concentrated with an Amicon Centricon filter (exclusion limit of Mr 10,000) and dialyzed against 10 mM Tris-HCl [pH 7.4], 50 mM NaCl, 20% glycerol and stored at −800 C. The eIF2α−ΔC protein was purified from E. coli as previously described [16 (link)]. Preparation of GST and GST fusion proteins of GCN2 were carried out as described previously [24 (link)].
7
Identification of Rqt1 Ribosomal Interactors
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To identify the putative target(s) of Rqt1, we co-expressed Myc-tagged ubiquitin (Myc-Ubi), which is partially resistant to proteasomal degradation55 (link), and the FLAG-tagged ribosomal protein uL23 (uL23-FLAG). Yeast cells harboring pCUP1p-MYC-UBI and pRPS2(uS5)-FLAG or pRPL25(uL23)-FLAG were cultured in 800 ml of synthetic complete medium. To induce the expression of Myc-Ubi, the cells were cultured in the presence of 0.1 mM Cu2+ for 2 h. Cell lysates were prepared and FLAG-tagged ribosomes were purified using M2 FLAG-affinity resin (Sigma), as described previously56 (link). Affinity purified samples were subjected to SDS-PAGE followed by western blotting with an anti-Myc antibody. The sections of the gels corresponding to the bands detected by western blotting were isolated and analyzed by mass spectrometry57 (link).
8
Assessing Ribosome Ubiquitination during ER Stress
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To assess ribosome ubiquitination during ER stress, ribosomes were purified with Myc-tagged ubiquitin (Myc-Ubi) and the FLAG-tagged ribosomal protein uL23 (uL23-FLAG), as described previously (Matsuo and Ikeuchi et al., 2017). Yeast cells harbouring pCUP1p-MYC-UBI and pRPL25(uL23)-FLAG were cultured in 800 mL of synthetic complete medium. To induce the expression of Myc-Ubi, the cells were cultured in the presence of 0.1 mM Cu 2+ for 1 h, followed by the addition of Tm to a concentration of 1 µg/mL and harvesting at the indicated time points.
Cell lysates were prepared, and FLAG-tagged ribosomes were purified using M2 FLAG-affinity resin (Sigma), as described previously (Inada et al., 2002). Affinity purified samples were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or western blotting with an anti-Myc antibody.
Cell lysates were prepared, and FLAG-tagged ribosomes were purified using M2 FLAG-affinity resin (Sigma), as described previously (Inada et al., 2002). Affinity purified samples were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or western blotting with an anti-Myc antibody.
9
Immunoblotting and Immunoprecipitation of Cortactin and AKT
10
NRF2 Purification and In Vitro Ubiquitylation
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Reactions were performed as described in Broderson M. M. L. et al. (2016), with the exception of NRF2 eluate from a PURExpress In Vitro Protein Synthesis Kit (NEB). Briefly, Flag tagged NRF2 was purified from HEK-293T cells with Flag-M2 affinity resin (Sigma) for Fig 2G or 250 ng of HALO-NRF2 was incubated with PURExpress components in a 25 μl reaction for 3 hours at 37°C for S4E Fig and 5 μl was used for each in vitro ubiquitylation reaction. CUL4 ligase (DDB1, CUL4A, WDR23) purification is described above.
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