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 37C, or
more vigorously at 34C.
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 -20C.


Day 2
3. Thaw the cells, add 5mgs lysozyme per litre of cells, room temp for 30 mins, add more PMSF.
4. Add 240l M MgCl2 and 24l M MnCl2 and 10-20l 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 -20C.



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


Supplies


Tips