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[…] Actin was purified from rabbit muscle according to () as modified by ().Expression plasmids for PPP1R15A (GADD34) variants contained ampicillin resistance marker, N-terminal GST tag and C-terminal maltose binding protein (MBP) tag (UK1677, UK1920)(). They were transformed into BL21 T7 Express lysY/Iq E. coli (C3013, New England Biolabs) and colonies that grew in LB-ampicillin plates (100 μg/ml ampicillin) were used to create a saturated over-night culture. This saturated culture was used to inoculate 2–4 Litres of LB media supplemented with 100 μg/ml ampicillin. The cultures were incubated at 37°C until OD600 = 0.6–0.8. At this point, they were induced with 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and cultured for 20 more hours at 18°C. It was followed by a centrifugation step to pellet bacteria and resuspension of the ice-cold pellets in 3–4 pellet volumes of lysis buffer (50 mM Tris pH 7.4, 500 mM NaCl, 1 mM MnCl2, 1 mM MgCl2, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 100 μM phenylmethylsulfonyl fluoride (PMSF), 20 mTIU/ ml aprotinin, 2 μM leupeptin, and 2 μg/ml pepstatin in 10% glycerol). An EmulsiFlex-C3 homogenizer (Avestin, Inc, Ottawa, Ontario) was used to lyse the bacteria, which were then clarified in a JA-25.50 rotor (Beckman Coulter) at 33,000×g for 30 min at 4°C. These suspensions were bound to pre-equilibrated glutathione sepharose 4B beads (17-0756-05, GE Healthcare) for 1–2 hr at 4°C. Beads were transferred to a 10 mL column after being batch-washed with 20 bed volumes of lysis buffer. Proteins were eluted in glutathione elution buffer (50 mM Tris pH 7.4, 100 mM NaCl, 40 mM glutathione, 0.5 mM MnCl2, 0.5 mM TCEP, 10% glycerol), and cleaved with Tobacco Etch Virus protease (TEV) (12.5 μg TEV protease/mg protein) overnight at 4°C to remove the N-terminal GST tag. Cleaved proteins were bound to amylose beads (E8021S, New England Biolabs) for 1–2 hr at 4°C. Twenty/thirty bed volumes of lysis buffer were used to batch-wash the amylose beads, which were transferred to a 10 mL column and eluted with HEPES buffer (20 mM HEPES, 100 mM NaCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM TCEP, 0.5 mM MnCl2, 100 μM PMSF, 20 mTIU/ ml aprotonin, 2 μM leupeptin, and 2 μg/ml pepstatin) and 10 mM maltose.PP1 (UK622) () was purified as above, with the following modifications: LB media cultures were supplemented with MnCl2, after TEV cleavage proteins were buffer exchanged using a 2 mL desalting column in HEPES buffer and re-bound to glutathione sepharose 4B beads to remove free GST tag.Phosphorylated eIF2α was encoded by an expression plasmid containing N-terminal His-Tag and kanamycin resistance marker (UK105) (). BL21 T7 Express lysY/Iq E. coli were co-transformed with this plasmid and a GST-Tagged PERK plasmid carrying ampicillin resistance marker (UK168)(). Colonies that grew in ampicillin (100 μg/ml) and kanamycin (50 μg/ml) LB-plates were used to create a saturated over-night cultured with which 2L of ampicillin and kanamycin LB were inoculated. Growth, induction and purification was as described for PPP1R15A, with the following changes: beads used were Ni-NTA (30230, Qiagen) to bind His-tag, lysis buffer contained 20 mM imidazole and elution buffer contained 500 mM imidazole instead of glutathione. This protein did not require TEV cleavage but an additional size exclusion chromatography step was included. A Superdex S200 (GE Healthcare) was used to gel filter the protein in 25 mM Tris, 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT and 10% glycerol buffer.All proteins were snap frozen and kept at −80°C in small aliquots. Final concentration of proteins was calculated from UV absorbance at 280 nm measurements in Nanodrop (Thermo Scientific, UK) and based on their extinction coefficient values predicted by MacVector. […]

Pipeline specifications

Software tools MUSCLE, MacVector
Application Nucleotide sequence alignment
Organisms Homo sapiens