Purifying Hemoglobin
Today we will try a new method to purify our protein
This will take place through FPLC using both a Q-Sepharose and a SP-Sepharose columns
- Spin down pH 10 protein from last week with 20mM Tris buffer at pH7.4
- This step is repeated three times at 3000rpm for an hour each time
- Make buffers for FPLC
- 1L 20mM Tris pH8.3
- 1L 20mM Tris with 1M NaCL pH8.3
- 1L 10mM Sodium Phosphate buffer pH 7.2
- 1L 10mM Sodium Phosphate buffer pH 8.0
- Equilibrate Q-Sepharose column with 20mM Tris buffer pH8.3
- Inject 10mL sample of protein into column
- Collect elute during injection
- Once loaded, run a gradient from 0 to 160mM NaCl in Tris pH 8.3 over 10 minutes
- This did not seem to elute anything, concentration of NaCl was risen to 300mM over 10 more minutes
- Fractions are collected during gradient
- These fractions were placed in a centrifuge filter with 50mL 10mM pH 7.2 phosphate buffer for 1 one hour at 4000rpm and 4C
- The initial and final elutes from the column (brown) were stored in the fridge along with the rest of the protein that wasn't used
- The SP-Sepharose column was attached and equilibrated with 10mM pH7.2 sodium phosphate buffer
|