Purified proteins were thawed and centrifuged at 20,000 x g for 10 min at 4°C to remove any potential aggregates. For 5mer runs, proteins were normalized to 1 μM prior to column loading. For 4mer + α runs, the 4mer was normalized to 1 μM and the α subunit was normalized to 1.2 μM. 100 μL of each sample was injected onto a Superose 6 10/300 column connected to an AKTA Pure 25 FPLC system (both from GE Healthcare). The system was run at 0.4 mL/min for 1 hr using 20 mM HEPES, 300 mM NaCl, 2 mM MgCl2, 1 mM TCEP, pH 7.4 as the mobile phase. For conditions with ISRIB, the protein samples and mobile phase were supplemented with 200 nM ISRIB.
Akta pure 25 fplc system
Manufactured by GE Healthcare
The AKTA Pure 25 FPLC system is a high-performance liquid chromatography instrument designed for protein purification. It is capable of automated sample handling, fraction collection, and real-time monitoring of the purification process. The system is suitable for a wide range of applications, including antibody purification, enzyme purification, and protein complex isolation.
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Lab products found in correlation
3 protocols using akta pure 25 fplc system
1
Size-exclusion Chromatography of Protein Complexes
2
eIF2B Complex Purification and Characterization
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Purified proteins were thawed and centrifuged at 20,000 × g, 10 min, 4 °C to remove any potential precipitate. Concentrations of eIF2B(βδγε) and eIF2Bα were normalized to 2 μM and 2.4 μM, respectively. 0.2 mL of each mixture was injected onto a Superose 6 Increase 10/300 column connected to an AKTA Pure 25 FPLC system (GE Healthcare). The system was run at 0.5 mL/min for 1 h using 25 mM HEPES, 200 mM KCl, 2 mM MgCl2, 1 mM DTT, pH 7.5 as the mobile phase. For conditions with ISRIB, the protein samples and mobile phase were supplemented with 500 nM ISRIB. For conditions with F6P, the protein samples and mobile phase were supplemented with 200 μM F6P. UV280 measurements were obtained directly by the instrument.
3
Purification of Recombinant Tetanus Toxin
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Recombinant TeNT was purified by NiSO 4 IMAC followed by size-exclusion chromatography (SEC) on an AKTApure 25 FPLC system (GEHealthcare). Briefly, post-expression, cells were pelleted at 4500 × g for 15 m. Cell pellets were either stored at -80°C or immediately sonicated on ice in IMAC buffer (50 mM Tris, 300 mM NaCl, pH 8). Post-sonication cell lysate was cleared by centrifugation at 4,500 × g for 15-30 m at 4°C and the supernatant filtered through a 0.45 μm membrane filter (Whatman). The sample, supplemented with 20 mM imidazole, was then applied to a 1 mL high purity Histrap NiSO 4 column (GEHealthcare) at 1 ml/min. The sample was washed and eluted with IMAC buffer supplemented with 20 mM and 300 mM imidazole, respectively. The eluted sample was concentrated, and buffer exchanged to phosphate buffered saline (PBS) using a Vivaspin20 column (50 kDa MWCO; Sartorious) as described by the manufacturer. Five hundred microlitres of sample was then applied to a Superdex 200 increase 10/300 size exclusion column (GEHealthcare) and eluted in PBS.
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