Clean and store Desalt column
- Run 150mL water through the column
- Run 150mL 20% Ethanol solution throughout the column
Make buffers for FPLC
- Prepare 400mL 20% Ethanol
- 80mL 200 poof ethanol in 320mL FPLC water
- Filter 1L H20 and prep for autoclave
- Prepare 1L 20mM Tris pH 8.3
- Filter and prep for autoclave
- Place buffer and water in autoclave at 121C for 1 hour
Prepare Protein solution
- Thaw tube on lab bench
- Place solution in centrifugal device with 30mL pH8.3 20mM Tris buffer
- Centrifuge for 30 minutes at 3000g
- Repeat above steps but this time for 1 hour
- Centrifuge until final volume is 10mL
Equilibrate Q-Sepharose column
- Run 25mL H2O through the column
- Run 25mL 20mM Tris pH 8.3
- Column is ready for protein
Purification fo Protein
- Inject protein into column
- Collect elute during injection
- Run a gradient 0-300mM NaCl in Tris over 10 minutes
- Collect fractions
- Combine desired fractions into single tube
- Clean column with 20mL 1M NaCl
- Re-equilibrate column with 20mM Tris pH 8.3
- Concentrate and desalt fractions using centrifugal device
- Add 20mM Tris to fractions twice to remove salt
- centrifuge until final volume is 10mL
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