Procedure
Concentration of the protein in CHES pH10 buffer was continued for another round
- 10mL of the concentrated solution was loaded in the FPLC
- The column was stabilized with 25mL of pH10 CHES buffer
- The protein was injected into the column
- At this point, the elute is being collected in a falcon tube
- Once the 10mL were loaded, 5mL of CHES buffer were run through to see if anything would come off
- A three step NaCL gradient was run through the column to separate compounds
- 0-200mM NaCl over 5 minutes
- Stay at 200mM NaCl for 3 minutes
- 200mM to 500mM over 5 minutes
- The peaks were observed in the third step but there seemed to be more than one compound contributing to the peak
- This was repeated by changing the third step to be 200mM-500mM over 20 minutes
- This did not produce the results we were hoping for
- There were no observable peaks
- The fractions were stored until a new method can be designed
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