|
|
Navigation: Go back to the Protein purification
Navigation: Go back to the Chapter 4. Protocols
Navigation: Go to: previous Analytical ammonium sulphate cut (AM cut)
Navigation: Go to: next Precipitation of proteins by ammonium sulphate
Protocols and tips in protein purification or How to purify protein in one day.
- Pour protein solution or crude extract in to a beaker with a magnetic bar in it. Choose a beaker volume 2.5-3 times larger than the volume of the sample.
- Place beaker in ice (or at 4°C) and on the stirrer. Set stirrer to mix solution with a speed which allows a vortex to form in the middle of the sample.
- If the first precipitating concentration is 2M (50% saturation) or lower of ammonium sulphate (this would be found during the analytical experiment), use 4M stock solution to bring the sample up to the desirable ammonium sulphate concentration. This is a much gentler way then using powder. Calculate the volume of the stock solution needed and add it to the beaker. Stop stirrer and incubate for 15 minutes.
- Spin down pellets in JA-20 or JA-25.50 rotor at 19000 rpm for 10 minutes.
- Measure the volume of the supernatant fraction and pour it back into the beaker that is on the stirrer (in ice bath or at 4°C). Check protein concentration to control the process.
- Using table, work out the amount of AS powder that is needed to precipitate the target protein.
- Weigh out the required amount of powder and add it to the sample (with stirring) in small portions, allow previous portion to dissolve before the next one is added. When all the salt has dissolved, turn off stirrer and leave sample for 15-30 minutes.
- Collect pellets by centrifugation as above, remove supernatant fraction completely and dissolve protein pellet in the appropriate buffer. Check volume and protein concentration to check loses.
NB: Remember that precipitation of protein depends on precipitant as well as protein concentration. Carry out the preparative and analytical experiments using the same protein concentration.
Remember: a significant concentration of ammonium sulphate is still present in the sample!
Navigation: Go back to the Protein purification
Navigation: Go back to the Chapter 4. Protocols
Navigation: Go to: previous Analytical ammonium sulphate cut (AM cut)
Navigation: Go to: next Precipitation of proteins by ammonium sulphate
|
