User:Paul Rothenberg/Notebook/Pauls Notebook/2014/04/02

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Protein Purification

  • Today, I ran the first part of protein reconstitution on the synthesized Mn PPIX myoglobin. The procedure for today is as follows:
  1. The column had ethanol present, and it was run out. The tip was also cut off.
  2. 15 mL of H2O was then run through it in 3x4 mL aliquots
  3. 25 mL of phosphate buffer was added to equilibrate the column
  4. Once the column was equilibrated, 2.5 mL of the reaction mixture was added, and the waste container removed
  5. 3.5 mL of buffer was added, and the product was collected
  6. 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:
  1. 0.0083 mol acid component
  2. 0.0116 mol basic component
  3. 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.