User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/09
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Preparation of more Apo-Mb
Andrew and I are going to synthesize more Apo-Mb for preparation of Co-Mb. We will follow the same protocol as I did here. We made a 0.05 M Tris solution, dissolved 200 mg of Mb into it, and added 40 μL of 1N HCl to bring the pH to 2.3. We then extracted with 4 mL of 2-butanone for two rounds.
We extracted about 7 mL of apoprotein. We also had to mix pH 2 H2O with equal parts 2-butanone because solid clumps blocked the separatory funnel. We added 4 mL of each for a total volume of 16 mL before separation. We added the apoprotein to a dialysis cassette and let run overnight in a 10 mM Sodium Bicarbonate buffer.
Purification of Co-PPIX
Andrew and I are going to continue filtering the Co-PPIX we synthesized last week. Once we have that, we are going to purify it and reconstitute the Apo-Mb we prepared today.