Hiprep 16 60 sephacryl s 200 hr gel filtration column

GST‐STAT3, Flag‐STAT3, His‐MST2, His‐JAK2, and mutant proteins were expressed in BL21(DE3) bacteria as previously described.¹⁴ Bacteria cells were cultured in Lysogeny Broth medium, and expression of these proteins was induced by IPTG for 16 hours at 30°C or 16°C, followed by lysis via sonication.
To purify the His‐MST2 and His‐JAK2, the lysed bacterial samples were transferred to a Ni‐NTA column (GE Healthcare Life Sciences). The column was flushed with 20 mM imidazole, and the protein was eluted with 250 mM imidazole.
To purify Flag‐STAT3, the lysed bacterial samples were transferred to a column containing Anti‐Flag M2 agarose affinity gel. The column was flushed with PBS and the protein was eluted with 100 μg/mL Flag peptide.
To purify GST‐STAT3, the lysed bacterial samples were transferred to a GSTrap HP column (GE Healthcare Life Sciences). The column was flushed with PBS, and the protein was eluted with 10 mM reduced glutathione.
To remove contaminated proteins, the eluted samples were separated through a HiPrep 16/60 Sephacryl S‐200 HR gel filtration column (GE Healthcare Life Sciences).

The DNA of WT and mutant G6PD was cloned into pCold I vector. The DNA of CK2α was cloned in to pGEX4T-1 vector. Recombinant constructs were transferred into BL21(DE3) bacteria as previously described [10 (link)]. Expression of recombinant proteins was stimulated by addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). BL21 bacteria cells were then cultured in Lysogeny Broth medium for 16 h at 16 °C or 30 °C, followed by lysis with sonication.
For the purification of His-tagged proteins, the lysed bacterial samples were transferred to a Ni-NTA column (GE Healthcare Life Sciences). The column was flushed with 20 mM imidazole and the protein was eluted with 250 mM imidazole. For the purification of GST-tagged proteins, cell lysates were loaded onto a GSTrap HP column (GE Healthcare Life Sciences), washed with five column volumes of PBS, and eluted with 10 mM reduced glutathione. To remove the contaminants, the eluted samples were separated through a HiPrep 16/60 Sephacryl S-200 HR gel filtration column (GE Healthcare Life Sciences).