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 column with the superloop's binding buffer flow at at 10 mL/min on inject. Then press end.
  • Sample 1 was loaded onto the super loop with the syringe at inject. The position was switched at position 3. The flow rate was at 5 mL/min with the gradient of B at 0%. From inject, the setting was switch to load.
  • The desired baseline should reach an absorbance of 280 and 250. Then hit pause.
  • The fraction is under the Frac 900 function in the dialog box. 5 mL fraction size was chosen. Then hit continue.
  • On the pump function, the load was set at 0% B after 25 mL the flow rate of 0 mL/min. was changed to 5 mL/min.
  • The same steps were done for sample 2.
  • The filtrate for sample 1 was collected from tubes 1-6 and sample 2 was collected from tubes 7-11.
  • Observing the graph below, the peak at fraction 2 indicates that most of the protein (ADA) was collected in this run. For sample 2, the protein filtrate was mostly collected at fraction 8.


Image:ADA HisTrapE.PNG


  • Abigail E. Miller 10:37, 7 October 2012 (EDT): did you not do anything else this day? I know you had atleast half the lab period left.





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