Glutathione sepharose 4b beads

The GST alone, GST-tagged and His-tagged proteins were purified from E.coli BL21 (DE3) system. The GST-tagged proteins were enriched by Glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions (Amersham Biosciences, Buckinghamshire, UK). His-tagged proteins were prepared and purified using Ni-affinity resins (Merk). His-PIM2 protein was mixed with GST or GST-HIF-1α (575-826aa) fusion proteins in PBS binding buffer (Takara's PBS, pH 7.4) at 4°C for 2 h, followed by the addition of 20 µl of Glutathione-Sepharose 4B beads. After 1h of mixing, the beads were washed with PBS five times. The pulled proteins were detected by western blots as previously described [22] (link), [23] (link).

Cells were lysed in a lysis buffer (Pierce, Rockford, United States). Lysates were then centrifuged and the supernatant was cleared by incubation with Protein A/G magnetic beads (Thermo Fisher Scientific) at 4°C temperature for one hour. The pre-cleared supernatant was then immunoprecipitated by incubation with the primary antibody SXT2 (ab3265, Abcam; 1:200) overnight at 4°C. Protein complexes were then incubated with Protein A/G magnetic beads for 1 h at 4°C and analyzed by western blot.
The proteins tagged with GST and His were amplified in Escherichia coli BL. Following the instructions, GST-tagged proteins were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for purification. His-tagged proteins were purified by nickel affinity resins (Merck). After adding 20 μm of glutathione-Sepharose 4B beads, the proteins tagged with His and GST or GST alone protein were mingled in PBS binding buffer (Takara’s PBS, pH 7.4, 4°C, 2 h) and then the mixture was cultured for 1 h with nutation. Finally, the beads were washed and eluted with 2x SDS sample buffer for western blot analysis.