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Purification of the NSP1 RD Protein
Posted on Tuesday, November 18, 2003
Description
Purification of the NSP1 RD Protein
Procedure
Day 1 1. Set up an overnight flask of 2 litres 2XTY Amp in a 2 litre flask, shake fairly gently at 37°C, or more vigorously at 34°C. 2. Harvest cells, resuspend in excess broth or 5-10mls of 50mM Tris pH 8.0, 25% sucrose, 1mM EGTA > 1mM PMSF and freeze at -20°C.
Day 2 3. Thaw the cells, add 5mgs lysozyme per litre of cells, room temp for 30 mins, add more PMSF. 4. Add 240µl M MgCl2 and 24µl M MnCl2 and 10-20µl DNase stand at room temp for 10-20 mins. 5. Spin at 20 K for 15 mins, remove and keep the supernatant, add more PMSF. 6. Dialyse the supernatant overnight vs 20mM imidazole pH 6.5,1mM EGTA,1mM DTT + PMSF. 7. Load the supernatant onto a CM column and wash in. Run a 0-150mM NaCl gradient, the protein elutes at around 35 mM Salt.or load onto a productiv CM column and elute stepwise, but this is not as good at concentrating the protein. 8. Dialyse the protein against 20mM Imidazole pH 7.0 50mM NaCl, 0.2mM EGTA, 0.2mM DTT + PMSF overnight.( optional, can load straight onto S100/S200)
Day 2/3 9. Concentrate in minicons 10K and load onto S100 or S200. The protein elutes in fractions 30-35 in 100 drop fractions.
Day 3/4 10. Concentrate in minicons, store at -20°C.
Recipes
PEG/TE/LiOAc • TE/LiOAc • DMSO • 2% glucose • 2% galactose • Dropout media • 2% raffinose • Deionized water • 25 x 75 x 1 mm frosted microscope slides • 24 x 40 mm micro cover slips • Phase contrast and fluorescence microscope
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