Q-Sepharose column
The remaining protein solution was placed in the fridge with 50mM Tris buffer at pH 8.3
- The Q-sepharose column was attached to the FPLC
- The protein solution was loaded in 50mL at a time
- the elute was collected during injection
- After each 50mL, a gradient of 0-160mM NaCl was run in Tris pH 8.3
- Fractions were collected during the gradient
- For the first 50mL, fractions 4-10 were collected
- For the second 50mL, fractions 15-21 were collected
- Fractions 1-3, 11-14 and 22-23 were collected in a separate tube for testing
- Between each injection, the column was cleaning with 1M NaCL and reequilibrated with 20mM Tris at pH8.3
- The column was cleaned and equilibrated with 20% Ethanol solution and stored
SP-Sepharose column
- The SP-Sepharose column was installed
- The filter to the FPLC was also changed
- The two sets of fractions were concentrated at 3680rpm at 4C in centrifugal devices with pH7.2 phosphate buffer 4 times in order to change the pH
- The SP-Sepharose column was equilibrated with pH7.2 phosphate buffer
- The two sets were loaded on the column and the run-through was collected
- The column was cleaned using 1M NaCl in tris pH8.3
- the elute was collected for testing purposes
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