- Today, I ran the first part of protein reconstitution on the synthesized Mn PPIX myoglobin. The procedure for today is as follows:
- The column had ethanol present, and it was run out. The tip was also cut off.
- 15 mL of H2O was then run through it in 3x4 mL aliquots
- 25 mL of phosphate buffer was added to equilibrate the column
- Once the column was equilibrated, 2.5 mL of the reaction mixture was added, and the waste container removed
- 3.5 mL of buffer was added, and the product was collected
- 25 mL of buffer was added to reequilibrate the column, and the procedure was repeated for the rest of the reaction mixture
- Two full 2.5 mL aliquots of protein were able to be run, and there was a third with under 2.5 mL of protein.
- 100 mM Phosphate Buffer, 200 mL:
- 0.0083 mol acid component
- 0.0116 mol basic component
- 200 mL H2O
- The procedure was run incorrectly, and the protein band did not separate out fully before the buffer was added. There was then a mixture of protein and buffer in the collection tube, and that must be reseparated in order to isolate and purify the protein.