A pGEX4T3-GST-RalBP1 expression vector, containing amino acids 397–518 of human RalBP1, was generated as previously described [7 (link)]. The vector was transformed in BL21(DE3)pLysS protease-free bacteria (Agilent Technologies, CA, USA) and grown to log phase in lysogeny broth media before induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoisde (IPTG) for 3 h at 30 °C. Bacteria were pelleted at 6000 rpm for 30 min (4 °C) in a Sorvall RC 6 Plus centrifuge (Thermo Fisher Scientific, SLA-1500 rotor) and lysed in 15 mL of Phosphate-buffered saline (PBS) lysis buffer (1 x PBS, 0.1% Triton X-100, 1 mM dithiothreitol (DTT), protease inhibitor tablet). The bacterial lysate was freeze-thawed, then supplemented with 100 μg/mL lysozyme before sonication, followed by lysate clarification at 12,000 rpm for 30 min (4 °C, Sorvall SS34 rotor). GST-RalBP1 was then purified by incubation with Glutathione Sepharose™ 4B (Thermo Fisher Scientific) for 90 min on a roller, followed by 3× washes in lysis buffer, 1 x wash in high salt buffer (0.5 M NaCl, 0.1% Triton X-100) and resuspension in storage buffer (1 x PBS, 50% glycerol, 1 mM DTT, 0.1% sodium azide).
Rc 6 plus
Manufactured by Thermo Fisher Scientific
The RC 6 Plus is a centrifuge designed for high-performance sample processing. It features a brushless induction motor and an ergonomic design for efficient operation.
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Lab products found in correlation
Glutathione sepharose 4b, by Thermo Fisher Scientific (1 mentions)
Imr 32, by American Type Culture Collection (1 mentions)
Neuro 2a, by American Type Culture Collection (1 mentions)
S5130, by Merck Group (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Sk n sh, by American Type Culture Collection (1 mentions)
Mt30002ci, by Thermo Fisher Scientific (1 mentions)
Hepg2, by European Collection of Authenticated Cell Cultures (1 mentions)
2 protocols using rc 6 plus
1
Expression and Purification of GST-RalBP1
2
Cell Culture Preparation and Delipidation
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The neuroblastoma cell lines Neuro-2a, SK-N-SH and IMR-32 were purchased from American Type Culture Collection. The hepatocyte carcinoma cell line HepG2 and lung carcinoma cell line A549 were purchased from the European Collection of Authenticated Cell Cultures (via Sigma-Aldrich). All cell lines were maintained as recommended by distributors, subcultured every 4–5 d up to 10 passages, and culture medium was changed every 2 days. All media components were purchased from Thermo Fisher Scientific Inc., including minimum essential medium eagle with glutamine (MEM, MT100010CV), Dulbecco’s modified eagle medium with glucose and sodium pyruvate without glutamine (DMEM, MT15013CV), L-glutamine (25-030-081), penicillin-streptomycin solution (P/S, MT30002CI) and N-2 Supplement (17-502-048). Premium grade fetal bovine serum (FBS) was obtained from VWR/Seradigm (97068-085). Delipidated FBS (dFBS) was prepared by stirring of silica (S5130, Sigma-Aldrich) into of FBS for 24 h at 4°C followed by centrifugation for 1 h at at 4°C in a Thermo Scientific Sorvall RC-6 Plus superspeed centrifuge. Supernatant was collected and validated for delipidation of cholesterol using UPLC-MS/MS derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD).
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