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1. Bacterium strain transformed with GST fusion protein gene plasmid DNA
2. Incubator shaker
3. Media: LB broth, NZY or Terrific broth containing 100 ug/ml ampercillin (1000x stock in -20oC refrigerate)
4. 50 ml culture tube
5. 250 ml flask
6. Eppendorf tubes
7. PBS
8. 1M IPTG
9. Glutathione beads (Sigma G4510)
10. Protease inhibitors stocks (PMSF, aprotinin, EDTA and leupeptin)
11. Triton X-100
12. 50mM Tris pH8/10mM GSH (sterile)
13. Sonicator
14. Lab..rotater
15. Bench top Centrifuge
16. spectrophotometer with 1ml cuvvett
17. protein gel and apparatus and reagents
18. (Optional: benzamidine beads)

a) Preparation of glutathione beads

1. Resuspend 1.5 g of glutathione beads (Sigma G4510) in 50 ml PBS
2. Swell 2h at 4 o C rotating.
3. Wash twice in PBS and resuspend in 1 bed volume of PBS at 4 o C.

b) Induction

1. Grow 10ml o/n culture from 100 ul glycerol stock in LB-Amp for a start culture.
2. Inoculate 5ml in 50ml NZY-Amp or Terrific Broth-Amp and shake at 37oC until OD 595 =0.7 (45-60min).
3. Add 50 ul 1M IPTG (1 mM final) and continue shaking for 3hr at 37oC (keep in mind that some proteins get insoluble or degraded if induction goes too long).
4. Spin @ 2000rpm 10 min (you will have about 450ul pellet),  resuspend pellet in PBS and transfer to a 15ml tube. Spin @ 3000rpm for 10min. Aspirate supernatant.
5. Resuspend pellet in 500 ul PBS containing 1% Triton and protease inhibitors
   

components	Stock	Volume in 1ml final
PBS	1X	850 ul
Triton      1%	10%	100 ul
PMSF     2mM	100 mM	20 ul
Aprotinin 100ug/ml	10 mg/ml	10 ul
EDTA     5mM	0.5 M	10 ul
Leupeptin 100ug/ml	10 mg/ml	10 ul

6. Vortex to disrupt pellet.
7. Cells are lysed by sonicating 3x15 seconds on ice (setting @ 4 - 6 ).
8. Transfer to 1.5 ml Eppendorf tube and spin 15 min at 4oC at top speed (14000rpm).
9. Decant supernatant ( Take 10ul supernatant + 4ul 5XSB+6ul H2O for gel analysis ) and add 250 ul glutathione beads (50% suspension).
10. Rotate 30 min at 4 o C
11. Spin @ 1000rpm, 1min ( Save super for gel analysis, 12ul super + 4ul 5XSB + 2ul H2O ) Wash beads twice in sterile PBS/triton buffer.
12. Wash twice in sterile PBS.
13. Elute by adding 250ul 50mM Tris pH8/10mM GSH.
14. Agitate 5 min at RT with shaker.
15. Spin 3min at RT.
16. Save supernatant and analyze by PAGE (4ul +4ul 5XSB + 12ul H2O) after removal of GSH
17. Add 125ul 2X sample Buffer to beads for gel analysis. (2ul + 4ul 5XSB + 14ul H2O)
    Heat mixture @95 o C for 5min and load 10 ul to 1mm 12% gel, run 1 hr @190V. Stain/destain and dry.
18. Removal of GSH from GST-NRG1 eluant with Centricon (refer to http://www.millipore.com/userguides.nsf/docs/P99259 )
    Transfer eluant into Centrifugal Filter Device (Millipore Centricon YM-10 (10k cutoff. Cat#4205, Fisher cat# ), ), dilute to 2ml with sterile PBS, put in 50ml centrifuge tube with cap. Spin 30min (or longer if needed until a small volume left) at 4000 x g with JA25-50 rotor in Beckman centrifuge. Add another 1-2ml PBS to remove GSH more efficiently. Spin. Repeate 2-3 times
    Collect GST-NRG1 by inverting unit onto retentate vial and spin 2-5min at 1000 xg.
19. Determine protein concentration by BCA reagent.
    
    

!!End for fusion protein preparation!!

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C. (Optional) Removal of GST Moiety from Fusion Protein

1. Combine 3 ml of bead suspension (50%) with fusion protein absorbed {from step B-12} , 10X thrombin buffer (Tris-HCl 8.0 500mM, NaCl 1M, CaCl2 25mM) {or PBS} and 150U thrombin (Sigma T1030). Shake 40 min at RT
2. Spin an d save supernatant. Analyze 15ul on minigel.
3. Add 500 ul GST beads to absorb any residual GST moiety. Rotate 1 hr at 4 o C
4. Remove supernatant after centrifugation and add 500ul of benzamidine beads prewashed in PBS (50% suspension; Pharmacia: BenzamidineSepharose 6B 17-0568-01 ) to remove thrombin rotating at 4 o C for 1 hr
5. Spin and save supernatant. Analyze 15ul by minigel.
   

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