https://eln.excellgen.com/index.php?action=history&feed=atom&title=RedB_Purification 2026-10-18T13:05:50Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=RedB_Purification&diff=3560&oldid=prev 2012-03-17T20:05:28Z

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

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