User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/26

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Purification of Proteins

  • Purification was achieved by running the collected filtrate through an affinity chromatography that is attached to a Fast Protein Liquid Chromatography (FPLC). The affinity utilized is the affinity of the His tags to nickel.
  • The initial step is to equilibriate the system by adding 50 mL of the clear, colorless binding buffer to the superloop using a syringe. Flowpath was at the generic position Load.
  • The end of pump A was inserted to the bottle solution of binding buffer; pump B was inserted into the bottle of the elution buffer. The pump was set at pump wash basic.
  • In consecutive order, the column was flushed with 25 mL of the binding buffer, 25 mL of elution buffer, and another 25 mL of binding buffer.
  • The flowpath was switched to position 3 after the run through of buffers. The pump flow was at 0.15 MPa and the flowrate was set to 5 mL/min.
  • From the readings, the detection indicates that the absorbance is at 280; indicative of the presence of Histidine. However, the absorbance at 280 is not exclusive to Histidine alone but also corresponds to Tryptophan, Tyrosine, and Phenylalanine.
  • The conductivity reading is sensitive to the amount of salt in the buffer.
  • On the dialog box, pump > gradient > B > 100%.
  • Once the peak levels back to baseline, pump > gradient > B > 0%. This washes the system with A.
  • At Flowpath > injection valve > position > inject. Switch to position 1. This bypasses the