User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/03
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Addition of Mn PPIX to Apo-Mb synthesized yesterday
Andrew and I are going to reconstitute the Mn-PPIX into the apo-Mb we prepared and left to run dialysis yesterday. For this, we are using the same protocol I ran here. Assuming a complete conversion of the protein, we have 500 mg of apoprotein in solution. Since it's 2 mg PPIX per 10 mg of apoprotein, we will use 50mg of PPIX because we did not run all of the apoprotein through dialysis. We have the rest in the fridge that we can use later.
The apoprotein was extracted from the dialysis cassette and placed in a beaker, and left to cool. Meanwhile, Andrew and I measured out 50 mg of Mn PPIX and dissolved it in 2 mL of autoclaved 100 μM Phosphate buffer. 10 drops of 0.1 N NaOH were added. The PPIX solution was added dropwise to the apoprotein, and left to react. The reaction was started at 10:00, and will react for 8-12 hours.
We also made 100 mM Phosphate buffer and autoclaved it for protein purification (from the last time I ran the reaction).
Synthesis of Co-PPIX
Andrew and I continued filtering the PPIX we made yesterday. We are going to filter it for another round to ensure maximum product yield from solution.