Last Update: December 2006
1. 1M IPTG (4 mL):
Mix 1 g of IPTG with 4 mL of dH2O (filter sterilize)
2. Cold buffer (200 mL):
Mix 10 mL of 1M Tris-HCl, pH 7.5, 6 mL of 5M NaCl, 400 mL of 0.5M EDTA, and 184 mL dH2O
3. 1X PBS (1 L):
Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, pH to 7.4 with HCl, and dH2O to 1 L
4. Wash solution (500 mL):
Mix 486.5 mL of 1X PBS, 12.5 mL of 20% Triton X-100 (0.5%), and 1 mL of 0.5M EDTA (1 mM)
5. 100 mM PMSF (10 mL):
Mix 170 mg of PMSF and 10 mL pf 2-propanol (Store at room temperature)
6. IB buffer (1 mL):
Mix in 1 mL of PBS, 1 mM EDTA, 0.1 mM PMSF, and 0.2 % sarcosyl
7. Wash I buffer:
1X PBS, 3 M NaCl
8. Wash II buffer:
0.1 M NaOAc buffer pH 4.5, 0.5 M NaCl
8. Wash III buffer:
1X PBS, 0.5 % Triton X-100
10. Fresh Elution buffer:
Mix 30 mL of 50 mM Tris-HCl pH 8, and 90 mg of Reduced Glutathione (10 mM)
1. In a 125 mL flask containing 10 mL of LB and 10 mL ofAmpicillin (100 mg/mL -for pGEX vectors, Life Technologies), transfer a single colony and Grow overnight
2. Transfer 5 mL of ON culture to a 250 mL flask with 45 mL of LB and 50 mL of Ampicillin (100 mg/mL)
3. Incubate at 28-37oC until A600 reaches 0.7 (best) - 0.8 (up to ~1.0). Induce with 10 mL of 1M IPTG/50 mL culture*. Incubate at 28oC (can also try at 37oC) for >2 hours at 250 rpms (longer incubations are OK if the GST-protein is stable and/or growing in BL21-DE3 proteolysis- cells)

*NOTE: varying the concentrations of IPTG may increase the yield of your protein (5 - 25mL of 1M IPTG/50 mL culture)

NOTE: growth of the expressed protein at 28oC after induction is supposed to reduce aggregation of the fusion protein
4. Centrifuge at 8,000 g for 10 mins at 4oC and discard the supernatant
Note: the pellet can be stored at -80oC for weeks
5. Resuspend pellet with 10 mL of cold buffer + 10 µL of 100 mM PMSF at 4oC.

BEST: in addition to the PMSF, other protease inhibitors should be added. A good cocktail of protease inhibitors is Complete (from Roche). This cocktail comes as a tablet that can be dissolved in 10 mL of buffer and provides good inhibition against 5 of the main proteases (at pH 7.8)

6. Lyze cells by 2-3 passages through a French press (4oC) at a pressure no lower than 1100 psi (keep pressure around 1100-1200 rpms) (cell lysis can be detected by clearing of suspension unless a high concentration of inclusion bodies is present)
NOTE: other means of cell lysis, like sonication, are not as good as a French press. In my hands, purifying the proteins I have purified, French-pressed samples yield more purified protein when compared to other extraction procedures. In a study I performed, 10 times more protein was obtained using a French-pressed sample as compared to a sonicated one

7. Add 0.5 mL of 20% Triton X-100/10 mL of lysate (to 1%Triton X-100). Mix on a rocker for 30-60 mins at 4oC (longer incubations may reduce the amount of inclusions bodies)

8. Centrifuge at 12,000 g for 20 minutes at 4oC and save the supernatant (can be stored at -80oC). This is termed total soluble protein = T

Optional: Inclusion Body (IB) proteins can be solubilized. Add 1 mL/50 mL culture pellet of IB buffer, sonicate (2-4x for 10-15 secs in a water bath somicator), resuspend and mix on a shaker for >30-60 mins at 4oC. Centrifuge at 12,000 g for 15 mins and save IB supernatant

9. Equilibrate a 50% slurry of Glutathione Sepharose 4B (Pharmacia Biotech, catalog # 27-4574-01) with PBS for 10 mins, spin for 2 mins at 500 g and discard wash. Add 10 mL of T / 2 mL of 50% slurry
Glutathione Sepharose 4B: store in 20% ethanol. 200-400 mmol Glutathione Sepharose per g dried substance. Spherical beads, 45-165 mm wet diameter. Spacer arm: 12 atoms (10 carbons)
10. Rock for >20 minutes at RT

11. Centrifuge at 500 g for 1 min. Save an aliquot of solution (Flow Through = FT)

12. Aspirate off supernatant withough getting too close to the beads and wash 4 times with 1X PBS/Wash solution each for <1 minute, namely invert the tube until the beads are resuspended (centrifuge for 2 mins at 500 g to pellet beads).
IMPORTANT: the affinity of the GST-fusion to the column will depend greatly on which protein has been fused to GST. In some cases the GST-fusion will associate so tightly to the column that long washes can be performed (5 mins) with little loss of protein. In most cases though, some fusion protein will be lost even during the washes, so reducing the wash time as well as using 1X PBS instead of the Wash solution should yield the highest amount of purified protein
13. Wash with 20 mM Tris-HCL pH 8.0 (optional)

14. Elute with 6 mL* of fresh elution buffer by mixing for 10 mins at RT. Consecutive elutions can be performed (E1 (~80%), E2 (~5%), E3). Store at -20oC

*NOTE: the beads can be transfered to a small 2 mL eppendorf tube and the elution carried out with only 1 mL of elution buffer in order to have a more concentrated sample. If this is done, consecutive elutions are more important as the first elution may not remove as much of the GST-fusion protein from the column
15. Regenerate column:

15.1 Wash with Wash I buffer and then with with Wash II buffer

15.2 Repeat this cycle 3 times

15.3 Wash 1X with Wash III buffer (this removes hydrophobically bound substances). A wash with 70% ethanol would do the same thing
15.4 Finally, re-equilibrate with 1X PBS

15.5 All washes are for 5 minutes. Washes are discarded after spinning down beads for 2 mins at 500 g

STORAGE: >1 month: wash one more time with 1X PBS and then repeat wash with 20% ethanol. Store at 4oC

Yield can be estimated by measuring absorbance at 280nm. For GST, 1 A280 = 0.5 mg/mL
Cross linking GST-Fusion to Sepharose-GSH resin

1. Follow the regular protocol as described above, after IPTG induction and centrifugation resuspend the cell with 10 ml of 20 mM Hepes-KOH, pH 7.8, 150 mM NaCl, 1 mM EDTA, and PMSF

2. French press, add 0.52 mL of 20% Triton-X-100, rock for 30 min 4oC, and centrifuge for 20 min at 12,000 g (4oC). Save supernatant (T); (T= Total soluble proteins)

3. Add supernatant (T) into a 15 ml tube containing 1.5 ml of GSH-sepharose resin (pre-washed with 20 ml 20 mM Hepes-KOH, pH 7.8, 150mM NaCl, 0.5% Triton-X-100)

4. Incubate at room temp for 20 minutes while rocking the tubes

5. Spin for 5 min at 1000 g, discard the supernatant

6. Wash the beads: Add 10 ml of wash buffer (20 mL 20 mM Hepes-KOH, pH 7.8, 150 mM NaCl, 0.5% Triton-X-100), rock for 5 min, spin for 2 min at 1000 g, discard the supernatant. Repeat washes 4 more times

7. In the last wash, remove as much supernatant as possible

Prepare the cross linker: 100mM DSS: Dissolved 18.4 mg of DSS (disuccinimidyl suberate: MW 368.3; Pierce - #21555) in 0.5 mL DMSO

8. Add 50-100 mL of 100mM DMS to beads, incubate at room temperature for 30 min followed by 1 h at 4oC. Every 5-10 min, mix by tapping tubes

9. Wash the beads once with 1 mL of wash buffer (save the wash) then wash twice with 10 mL of wash buffer as in step 6

Binding of antibody

1. Pre-adsorbtion: incubate 1-2 mL of antibodies (prepared against GST-fusion) with 500 mL of beads (that were cross linked to GST) for 2 h in the cold room while rocking

2. Spin for 5 min at 1000 g. Remove as much of the supernatant and save

3. Incubate the supernatant antisera with 1.5 mL of beads cross-linked to GST-fused to your protein for 2 h or over-night in the cold-room on a rocker

4. Spin for 5 min at 1000 g, remove as much of the supernatant and save to be analyzed later

5. Wash twice with 10 mL of wash buffer as above

6. Elute the antibodies: add to the beads 1 mL of 0.1M glycine-HCl pH 2.5, incubate at 4oC for 15-20 min on a rocker

7. Spin for 5 min at 1000 g and remove as much of the supernatant containing "pure-AB"

8. Add to the pure Ab solution (1 mL) about 22 mL of 2M Tris base to bring the pH to ~7

9. Add to the beads an additional 0.2 mL of glycine-HCl, incubate for 5 min, spin and save the supernatnat. Add Tris base. Combine both eluitions

10. Test your Pure Ab on western at several dilutions


Preparation of GST-fusion protein

1. Prepare Total E.Coli lysate (induce with IPTG, French press, centrifugation)

NOTE: total lysate can be stored at -80oC until use. As control used GST alone

2. In eppendorf tube (1.5 ml) bind 0.5 mL of total lysate (~1-3 mg of GST-fusion protein) to 100 ml of 50% slurry of GSH-sepharose beads previously equilibrated with 6 mL of 20mM Tris-HCl, pH 7.5; 1mM Mg(OAc)2; 150 mM NaCl; 0.1-0.5% Triton-X-100

3. Centrifuge for 2 min at 500 g, discard flow through (save 50 mL for later analysis)

4. Wash the unbound proteins 4 times each with 2 mL of 20mM Tris-HCl, pH 7.5; 1mM Mg(OAc)2; 150 mM NaCl; 0.5% Triton-X-100

5. If protein fused to GST needs to be activated prior binding to plant extract: add to 3 mL of bound GST-protein:

5.1 I - GTP to a final concentration of 1mM

5.2 II - 1mM GDP

5.3 III -1mM ATP

5.4 IV - 1mM GTP

Incubate at cold room for 15 min while rocking, then add transfer the Total into a 2 mL ependorff tube containing 50 mL pre-washed GSH-sepharose beads, incubate for 10-20 min to allow binding of the activated protein fused to GST

6. Wash proteins 1 time each with 2 mL of 20mM Tris-HCl, pH 7.5; 1mM Mg(OAc)2; 150 mM NaCl; 0.05-0.1% Triton-X-100

7. The sepharose bound GST-protein is ready (stored on ice)

Affinity chromatography

1. Mix 0.5 mL of S1 plant extract pre-treated with or w/o 1 W/O

2. Mix 2 mL of S13 plant extract pre-treated with or w/o:

2.1 50mM GTP - or GTP gS

2.2 50mM GDP - or GDP bS

2.3 50mM ATP - or ATP gS

2.4 50mM ADP

2.5 50mM UTP

Preparation of plant crude extract

1. Incubate for 20 min at room temperature, or 4oC, while rocking

2. Centrifuge at 500 g for 2 min at 4oC. Discard the flow through FT (save a 0.2 mL aliquot)

3. Wash the unbound proteins 4 times each with 1.2 mL of 20mM Tris-HCl, pH 7.5; 1mM Mg(OAc)2; 0.05-0.1% Triton-X-100. Save the last flow through LFT

4. Elute the GST-fusion protein + interacting plant proteins from the agarose beads by adding 100 mL of elution buffer, mix at room temperature for 20 min, centrifuge for 5 min at 500 g, collect the eluted protein (= EL1). Add to the beads 100 mL more of elution buffer, mix for 10 min, centrifuge and save eluted protein (= EL2) (Over 70% is eluted in EL1 fraction)

5. Column regeneration: Wash with 50 mL PBS+3M NaCl; then 0.1M acetate buffer pH 4.5, 0.5M NaCl, followed by PBS, then PBS+0.5% Triton X100. The column resin can be used up to 5 times