Purifying hemoglobin
- Collect frozen protein solution from last week and thaw
- Once thawed, the solution's pH was changed using concentration device for centrifuge
- 15mL of protein solution was added to 45mL of pH8.3 50mM Tris buffer
- The solutions were spun at 3000rpm for 30 minutes
- Repeat the last step
- The solutions were spun until the final total volume was less than 40mL
- Spin down concentrated protein solution to remove impurities
- Equilibrate Q-Sepharose column with 50mM 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 collected in 5mL fractions
- A peak was collected in this range
- tube labelled fractions 11,12,13
- concentration is raised to 300mM
- Another set of fractions is collected and placed in tube labelled fractions 14-23
- These fractions were placed in a centrifuge filter with 50mL 10mM pH 7.2 phosphate buffer for 1 one hour at 3500rpm and 4C
- Repeated twice keeping the two sets of fractions separate
- 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
- 10mL of each set of fractions were loaded separately
- The run-through was collected in tubes labeled with the corresponding fractions
All solutions stored in fridge overnight
|
Project name
