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Navigation: Go back to the Protein purification
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Protocols and tips in protein purification or How to purify protein in one day.
- When harvesting cells from culture media spin down cells first in the big bottles, than re-suspend pellets in about 50 ml of culture medium, place in 50 ml Falcon tube and spin down for 10-15 min at 5000 rpm. Remove medium and put tube with cell pellets in to the -20°C freezer.
Alternatively suspend cells in buffer A (50 mM tris pH 8.0) (5-10 ml per gram of cell paste) and put it in to the -20°C freezer.
- On the day of protein purification take cell paste from the freezer, add 8-15 ml of buffer A per gram (ml) of cell paste, let it thaw for 5 minutes, briefly suspend cell paste with spatula and divide suspensia into 15 ml portions placing them into 20 ml plastic vials. If cells were frozen with buffer, for quick defrosting place tube in to warm water or hold under a hot water tap, mixing by tipping upside down and right way up again until it thaws. Then divide into portions as above. Place vials in an ice bath.
Tip: for more effective cooling put small pieces of ice in to the vials.
- Before you start sonication, put rotor (JA-20 or JA-25.50) into the Avanti centrifuge, close the lid and set pre-cooling at 4°C
- Mount medium probe on a Soniprep 150 machine. Tighten it properly with the tool, but do not over force. Lower probe in to the vial with the sample, leaving 2 mm between the probe and the bottom of the vial, the probe should not touch the bottom of the vial but neither should it be close to the surface of the sample. If the probe is too close to the surface, foam formation would occur, this should be prevented to avoid oxidation and denaturing of proteins.
- Set sonicator for maximum force. This corresponds to 16 micron amplitude. Sonicate portions one after another for 20 sec each. Carry out 3/4 cycles without a break. The samples would cool down while you treat other portions.
NB If power does not reach 16 micron, try reattaching the probe (you may need to tighten it a little more).
- After completion of sonication unscrew the probe, wash it with distilled water and dry with tissue. Place back in the cupboard.
- Pour homogenate in to the centrifugation tubes, balance them and spin down in JA-20 or JA-25.50 rotor at 4°C
for JA-20 rotor in J-20 centrifuge set 19000 rpm (about 43000g) for 15 minutes
for JA-25.5 rotor (red) in J-25 Avanti centrifuge set 24000 rpm (about 70000g) for 10 min
- Once centrifugation has finished pour supernatant fraction into the cylinder and keep it cold. Check volume. Check protein concentration (see protocol for Bradford assay in Appendix). Calculate total protein.
The Crude extract is now ready for the next step.
Navigation: Go back to the Protein purification
Navigation: Go back to the Chapter 4. Protocols
Navigation: Go to: previous Preparation of the stock solutions
Navigation: Go to: next Protamin sulphate treatment
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