Glutathione sepharose slurry

Fragments of bbYY1 or hsYY1 were inserted into the pGEX-6P-1 vector for GST-fused protein constructions. The constructs were then transformed into E. coli. BL21 bacteria. The transformed bacteria were cultured at 37 °C to an OD = 0.6–0.8, chilled to 18 °C and induced with 0.5 mM IPTG. After 12 h of induction, the cells were collected, resuspended, and sonicated with lysis buffer (TBST with protease inhibitor cocktail). The lysate supernatant was incubated with 40 μL of Glutathione-Sepharose slurry (cat.: 17-5113-01, GE) for 1 h at 4 °C to pull down GST-fused protein.
The MBP-bbRAG1L/2L expression vectors were co-transfected into 293 T cells for MBP fusion protein preparation. The transfected cells were continually cultured 60 h after vector transfection and lysed using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). The lysate supernatant was rotated with 40 μL of the above well-prepared slurry (containing GST or GST fusion protein) in TBST buffer (with protease inhibitor cocktail addition) at 4 °C overnight and washed four times with TBST. Finally, the slurry binding with proteins was eluted in Laemmli buffer (supplemented with 200 mM 2-mercaptoethanol) and resolved on 10% SDS-PAGE gels for further western blotting analysis. The ECL chemiluminescence method was used for the western blot analysis.

Recombinant GST-conjugated PDK1 was generated by transforming the BL21 (DE3) E. coli strain with pGEX-4T1-PDK1 or pGEX-4T1-1 (Empty vector control). Starter cultures grown overnight at 37 °C were inoculated (1%) into larger volumes. Cultures were grown at 37 °C until an O.D. 0.8, following which protein expression was induced for 16 h using 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 °C. Pellets were re-suspended in EBC buffer and sonicated. The supernatant was incubated with Glutathione-sepharose slurry (GE) for 2 h at 4 °C. The Glutathione beads were washed 3 times with PBS buffer and stored at 4 °C in EBC buffer or eluted by elution buffer and further analyzed by Coomassie blue staining and quantified by bovine serum albumin (BSA) standards.
The recombinant His-PDK1 was generated by transforming the BL21 (DE3) E. coli strain with by the same strategy as GST-tagged proteins. The difference was that the supernatant was incubated with Nickel resin slurry (Qiagen) for 2 h at 4 °C. The Nickel resins were washed 4 times with Tris-buffered saline (TBS) buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl) containing 10 mM imidazole (Sigma) and eluted by TBS buffer containing 100 mM imidazole.