User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/18

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Reconstitution of Apoprotein

Going to reconstitute the Apoprotein with MnPPIX. Hopefully the yield will be greater today. Before that, I ran a UV-VIS spec of the apoprotein at 1/20 and pure to compare concentration values. It looks like we are losing product during dialysis.

Results of Dialysis

Apo Mb Post Dialysis June 18.png

Procedure for Today:

To synthesize more apoprotein, we made a stock solution of 50 mM Tris buffer at pH 7.2. We dissolved 50 mg of myoglobin into 20 mL of buffer, and placed it on ice. The pH was brought to 2.5 at 0°C with the addition of 3.2 mL of 1 N HCl. Once the HCl was added, the solution was immediately mixed with equal volume of 2-butanone, shaken for 30 seconds, and let sit for 1 minute. Three total extractions were performed, and about 25 mL of apoprotein extract was obtained. The pH of the 2.5 solution was tested, and found to be below 2. See Andrew's notebook for more details.

For dialysis, the pH 2.5 sample has no orange clips. The pH 3.0 sample has two orange clips, along with the two green clips.

Results of Second Extraction

Apo Mb Extraction 2 Graph.png