User:Paul Rothenberg/Notebook/Pauls Notebook/2014/06/16
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After lyophilizing the Mn Mb last week, we found that there was only 2 mg total produced (out of 200 mg of protein!). We restarted, so we synthesized more ApoMb. We doubled the volume of the Tris buffer to 8 mL with the same concentration, and only used 100 mg of protein, not 200 mg. We are going to be more cautious and ensure that the protein is fully in solution before performing extraction. We then centrifuged the solution at 4,000 rpm for 30 minutes at 25°C before the extraction (should've been run at 4°C. Next time).
We brought the pH down to 2.3 with 40 µL of 1 N HCl, and did the acid/2-butanone extraction. We ran a UV-VIS spectrum for both extracted layers. The aqueous layer was highly concentrated (>>1.0), so a series dilution was performed. 10 µL of the apoprotein was diluted with 190 µL of H2O. This was still heavily concentrated; so a further series dilution was performed. A 1/20, 1/400, and 1/2000 dilution series was performed. There was no peak at 400 nm, so there was no noticeable amount of heme in the sample.
The collected organic layer was filtered and the solid collected and resuspended in H2O/50 mM Tris buffer. It was centrifuged for 5 minutes. The tubes spilled out when the centrifuge finished, so no UV-VIS spectrum could be taken.