Editing RedB Purification

== Protocol 1 ==

1. Grow pBD24-RedB in BL21-AI (AI: Arabinose inducible) at 37 °C.<br>2. Dilute overnight culture 1/50 into 1 liter of LB-Amp, grown to an OD600 value of 0.5.<br>3. Induce with 0.2% (w/v final concentration) of L-Arabinose for 4 hours of induction.<br>4. Spin down cells, resuspend in 35 ml of resuspension buffer (50 mM Tris-HCl pH 7.5, 10% sucrose).<br>5. Store cell pellet at -80 °C.<br>6. Thaw frozen cells at room temperature.<br>7. Sonicate or homogenize lysate. <br>8. Spin at 12000 rpm for 20 min.<br>9. Collectsupernatant, add polyethylenemine dropwise to a 0.5% (v/v) final concentration while stirring. Stir for another 30 min.<br>10. Spin at 35,000 rpm for 15 minutes at 4 °C. Discard supernatant.<br>11. Extract pellet with extraction buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 500 mM NaCl, 1 mM DTT, 10% Glycerol).<br>12. Spin the solution at 12,000 g for 45 minutes.<br>13. Collect supernatant, add solid ammonium sulphate slowly to 45% (w/v) final concentration while stirring. Let it sit overnight in a cold room.<br>14. Spin at 12,000 g for 15 minutes.<br>15. Resuspend the pellet in 35 ml nickel-column BufferA (200 mM NaCl, 20 mM Tris-HCl pH 7.6, 5 mM imidazole), spin at 20,000 g for 30 minutes.<br>16. Load supernatant into a nickel affinity column.<br>17. Elute with a 5 mM – 500 mM imidazole gradient in BufferB.<br>18. Add ammonium sulphate slowly to fractions containing Redβ, to 60% (w/v) final concentration, let sit 30 min.<br>19. Spin 20,000 g for 30 minutes. <br>20 Resuspend the pellet in 10 ml MonoQ-A buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 1 mM DTT, 5% Glycerol, 75 mM KCl), and dialyze against the same buffer (500 ml) overnight. <br>21. Spin to remove insoluble material. Load supernatant to a MonoQ column, <br>22. Elute with a 75 mM-450 mM gradient of KCl. <br>23. Dialyse fractions with storage buffer (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 1 mM DTT, 50% Glycerol) overnight at 4 °C. <br>

[[Category:Protocols]]