Purification of Caspase 9 protein

10/13/99

DAY 1

1. Freshly transform constructs in PRSET vector into BL21(DE3)pLysS bugs (Stratagene catalog # 200132)

50uL bugs on ice to thaw

add 0.85uL BME (supplied with bugs) to each tube, 10’ on ice

add DNA (they say 1-50mg, I use about .5-1ug), 30’ on ice

Heat shock 45sec at 42°

Add 0.5 mL LB and shake at 37° for 1hour

Plate full transformation on an LBA plate (gives about 100-200 colonies)

DAY2

2. Pick single colony and grow 20-50mL O/N in LB plus Amp.

DAY3

3. Dilute O/N into 500mL warmed LB plus Amp.

4. Grow until OD600 = 1.0 (can take up to 5 hours!)

5. Induce with 0.4mM IPTG (2mLof 100mM/500mL culture)

6. Grow for 2 hours

7. Take small aliquot for gel

8. Spin down bugs in GS-3 rotor, 8000g for 10’

9. Discard LB

10. Resuspend pellet by scraping/vortexing in 50mL lysis buffer plus protease inhibitors.

11. freeze O/N –20°

DAY4

12. Run test gel for expression in the induced bugs

10% gel, C9 is at 45kD, anti FLAG 1:1000, G anti mouse

DAY 5

13. Thaw tubes and sonicate 3 x 1’ on ice

14. spin 30’ in falcon 50mL tubes, 9000RPM GS-3

15. start resin preparation – use 3mL 50% slurry for each prep. (Invitrogen “Probond” resin) Wash 2X 7mL ddH2O, 3X native binding buffer. Spin out resin in clinical centrifuge.

16. spin 30’ in oak ridge tubes, SS34, 12K

17. filter supe through .45um filter

18. Add 3mL nickel resin 50% slurry in native binding buffer. Rock for 60’ at 4°.

19. Spin down beads 5’ in cold room clincal fuge.

20. Wash 3X 20mL Native binding buffer

21. wash 2X 10mL native wash buffer. Pour last wash on BioRad column.

22. elute with 3mL 50mM imidazole – allow to flow through

23.elute with 1.5mL 200mM imidazole, then 1.5 mL 350mM imidazole. Collect together. Take sample for protein assay and for gel to test recovery.

24. Inject 3mL sample into 10K cutoff slidalyzer (Pierce). Diazlyze O/N at 4°C, 1L per sample.

DAY 6

25. Run test gel to assess protein recovery

26. Withdraw small sample to repeat protein assay.

27. Change to dialysis solution with protease inhibitors and dialyze for 4 more hours.

28. Withdraw purified protein, place in small aliquots and store at –80°C.

Recipes:

Lysis buffer:

5mM imidazole 1.1mL 2.27M

500mM NaCl 50mL 5M

20mM Tris pH 7.9 10mL 1M (pH 8.0)

0.1% Tx-100 2.5mL 20%

416.4mL ddH2O

add fresh:

5mL PMSF, aprotinin, leupeptin, pepstatin

Native Binding Buffer:

20mM sodium phosphate pH 7.8 20mL 0.1M

500mM sodium chloride 10mL 5M

70mL ddH2O

0.1M sodium phosphate pH 7.8:

89.6mL 1M Na2HPO4

10.4mL 1M NaH2PO4

in 1L

Native Wash Buffer:

20mM sodium phosphate pH 6.0 20mL 0.1M

500mM sodium chloride 10mL 5M

70mL ddH2O

0.1M sodium phosphate pH6.0

12.0mL 1M Na2HPO4

88.0mL 1M NaH2PO4

in 1L

Elution buffers:

2.27M imidazole wash

50mM 0.11 4.98

200mM 0.43mL 4.57

350mM 0.76 4.24

Dialysis solution:

20mM Tris pH7.5 20mL 1M (or 2.42g Tris base pH to 7.5)

250mM NaCl 50mL 5M (or 14.61g)

1mM EGTA 2mL 0.5M

in 1L

second dialysis:

add 0.03% NP40 0.3mL

2mM DTT 2mL 1M

0.1mM PMSF 1mL 0.1M

IPTG 100mM

238mg in 10mL ddH20

sterile filter and store at -20