Rab 5 from BL21

Lysis buffer (100ml)

50mM Tris, pH 8

1mM EDTA

10mM mercapto ethanol

10µM GDP

add inhibitor cocktail prior to use.

Salty lysis buffer

50ml lysis buffer, add NaCl to 0.3M, MgCl2 to 10mM

NTA wash buffer

50mM MES-NaOH, pH 6

0.3M NaCl

1mM MgCl2

50µM EGTA

10mM mercaptoethanol

10µM GDP

1. 5ml overnight cultue

2. 5ml-> 1 litre LB, incubate until OD=0.8

3. Add IPTG to 0.4mM, incubate for 3 hours.

4. pellet and wash with PBS.

5. Resuspend in ~30ml lysis buffer

6. Snap freeeze and store or

add lysozyme to 0.4mg/ml (lysoztme is stored as apowder in -20° freezer, G.08 (molecular biology general enzymes box).

freeze and thaw x2.

7. Add NaCl to 0.3M, MgCl2 to 10mM.

8. Add 10µg/ml DNAse, incubate on ice for 30 minutes with gentle stirring.

9. Pass through 23G needle X3.

10. Centrifuge 33,000rpm (kontron, 55.38) for 45 minutes.

11. Prepare Pharmacia His-trap column- wash with 5ml water-> 3ml 0.1M NiCl2-> 5ml water -> 5ml NTA wash buffer -> 5ml NTA/250mM imidazole(NTA/250)-> 5ml NTA wash buffer, all using 5ml syringe.

Apply sample and collect flow thru and subsequent fractions.

Wash 5ml NTA wash buffer

-> 5ml NTA/20

-> 5ml NTA/50

->5ml NTA/100

->5ml NTA/250

regenerate column according to manufacters instructions.

12. Run 12% gel, rab5 starts coming off in NTA/100

but larger and purer fraction with NTA/250

13. Dialyse NTA/250 fraction overnight against 2x1.25 litres

50mM Tris-HCl, pH 7.2

1mM MgCl2

50µM GDP