20a-hydroxysteroid

E. coli transfected with pGEX-4T-3-20?HSD was grown in 2 × YTG medium. 1PTG was added to induce the expression of fusion protein. Twenty micrograms of protein were used as a starting material. GST and rec GST-20?HSD were purified with glutathione beads. To release 20?HSD, the bound GST-20?HSD proteins were digested with thrombin at room temperature for 6 h. The purified rec GST-20?HSD and purified rec 20?HSD proteins ( ?g each) were subjected to SDS-PAGE, transferred to cellulose membranes, and immunodetected with the anti-20?HSD antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit IgG. Left panel, Gel stained for proteins with Coomassie blue. Right panel, Immunoblots. Lane 1, Purified GST; lane 2, purified GST-20?HSD; lane 3, thrombin cleavage for fusion protein bound to glutathione-Sepharose 4B; lane 4, proteins remained absorbed to column after thrombin digestion.

20a-hydroxysteroid

20a-hydroxysteroid

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