Journal: Methods (San Diego, Calif.)

Article Title: Reconstitution and Analyses of RNA Interactions with Eukaryotic Translation Initiation Factors and Ribosomal Preinitiation Complexes

doi: 10.1016/j.ymeth.2019.03.024

Figure Lengend Snippet: eIF3 purification from yeast. Lysate from cells overexpressing His-tagged eIF3B were fractionated on Nickel, Phosphocellulose (P11), and Superdex 200 columns to purify the core eIF3 complex. Chromatograms are shown for Nickel and Superdex columns, with A260 and A280 traces shown in red and blue, respectively and percentage buffer B (high imidazole) in orange. 10% SDS-PAGE gels stained with Coomassie are shown. The gel run following P11 (center) shows 1) clarified lysate, 2) MW marker, 3) P11 bound, 4) Ni eluate/P11 load, 5) P11 flowthrough, 6) P11-B200 eluate, 7) P11-B350 eluate. Asterisks mark contaminants that are consistently found in B350/1000 fractions. The larger contaminant is associated with RNase activity and the lower is a similar size to eIF3 ligands eIF5 and eIF2-gamma. The lower gel shows the final purified protein next to MW marker (Precision plus, Biorad: 250, 150, 100, 75, 50, 37, and 25 kDa bands), with the subunits and approximate MWs indicated.

Article Snippet: BL21-CodonPlus (DE3)-RIL Competent Cells (Agilent #230245) pET21c-TIF1 plasmid (Addgene #37230) LB Carbenicillin agar plate 250 ml flask with 50 ml and 6 × 2.8L Fernbach flasks with 1.5L LB containing 50 μg/ml Carbenicillin and 34 μg/ml Chloramphenicol (LB Carb/Cm) 10% SDS PAGE materials and Coomassie stain HiTrap Q HP column (5 ml, GE #17115401) Superdex 200 increase 10/300 column (GE #28990944) CHT Type II Hydroxyapatite column (Biorad #7324332) Ammonium sulfate 0.45 μm PVDF syringe filters 2 M MgCl 2 Benzonase nuclease (Sigma, ≥250 U/ml) 0.1M NaOH Lysis Buffer: 20 mM HEPES-KOH (pH 7.4), 150 mM KCl, 0.1 mM EDTA, 10% (v/v) Glycerol, 2 mM DTT*, 1 Roche Complete EDTA-Free tablet/50ml* Q50 Buffer: 20 mM HEPES-KOH (pH 7.4), 50 mM KCl, 0.1 mM EDTA, 10% (v/v) Glycerol, 2 mM DTT* Q1000 Buffer: 20 mM HEPES-KOH (pH 7.4), 1 M KCl, 0.1 mM EDTA, 10% (v/v) Glycerol, 2 mM DTT* Low Phosphate Buffer: 5 mM Potassium Phosphate, pH 6.8, 150 mM KCl, 10% (v/v) Glycerol, 2 mM DTT* High Phosphate Buffer: 500 mM Potassium Phosphate, pH 6.8, 150 mM KCl, 10% (v/v) Glycerol, 2 mM DTT* Enzyme Storage Buffer: 20 mM HEPES-KOH, pH 7.4, 250 mM KOAc, 10% (v/v) Glycerol, 2 mM DTT* *Add Protease Inhibitors and/or DTT to buffers just before use Note: Buffers are kept at 4°C unless otherwise specified.

Techniques: Purification, SDS Page, Staining, Marker, Activity Assay