Research Protocols

Here are a few protocols that may be of some help .....

Purification of GST fusion proteins

Grow ~200mL overnight culture of bugs containing desired fusion construct. Inoculate 4 x 1.5 LB/Amp (for total 6l0 with ~50 mL of the overnight.
Grow cultures at 37C to OD600 ~ 0.6.
Add IPTG to final concentration of 0.25mM and continue to incubate at 37C (or 18-30C for highly insoluble protein) for 2h-4h to induce fusion protein (or O/N for low temp.).
Harvest cells by centrifugation, resuspend in a small volume of PBS (keep cells on ice during resuspension) and pellet in CSA bottle.
Freeze pellets in liquid nitrogen. Frozen pellets may be stored at -80C.
Resuspend frozen pellet in ~5 volume PBS+lysozyme. EGTA, EDTA and protease inhibitors (see below).
Resuspend either by pipetting up and down or by stirring on a magnetic stirrer (in a cold room) or both.
Pass through the Avestin homogenizer 3 times at 12-20,000psi. Keep lysate on ice at all times.
Add KCl to final concentration of 0.25M and DTT to 15mM. Spin lysate 1h at 40K in Beckman 70Ti rotor. Collect supernatant.
Load supernatant from 7 onto ~5-10mL glutathione agarose column equilibrated in PBS + 0.25 M KCl + 0.5 mM DTT (this is buffer A if using FPLC) at flow rate of 0.3 mL/min.
If time permits, re-load flow-through onto column (binding is rather inefficient).
Wash with the same buffer until A280 is flat, baseline.
Elute bound proteins with buffer B: 50mM Tris-Cl, pH 8.0, 250 mM KCl, 5mM glutathione. Collect 1mL Fractions and check by SDS-PAGE.
Pool peak fractions and dialyze extensively against dialysis buffer C (3-4 changes, 100 volumes each, over 4-6h).
Measure protein concentration. Make aliquots and freeze in liquid nitrogen. Store at -80C.

Glutathione-Agarose (Sigma G-4510)
0.25M reduced glutathione in water
20mg/mL lysozyme stock
70mM Na2PO4 19.9g
68.2mMNaH2PO4 18.9g
1.8mM KH2PO4 0.489g
138mMNaCl 16.2g
2.7mMKCl 0.403
PBS + lysozyme, DTT, protease inhibitors
Same as PBS above + the following components, added just prior to use:
1mg/mL leupeptin
2mg/mL aprotinin
200mg/mL lysozyme

PBS + 0.25 M KCl, 0.5 mM DTT (Buffer A)
Same as PBS above +
0.25M KCl
0.5mM DTT
Elution Buffer (Buffer B) 250mL
50mM Tris-Cl, pH 8.0 12.5mL 1M
O.25M KCl 4.66g
5mM glutathione 384mg
Dialysis Buffer (C) 2L
50mM Hepes, pH 7.6 100mL 1M
50mM KCl 7.46g
30% glycerol 600mL
1mM DTT 2mL 1M

Metal Chelate Affinity Chromatography

Phosphate Buffer A
50mM Na-PO4 pH 8.0 46.6mL 1M Na2 PO4 + 3.4mL 1M NaH2PO4
300mM NaCl 60mL 5M
10% glycerol 100mL

Phosphate Buffer B
50mM Na-PO4 pH 8.0 46.6mL 1M Na2 PO4 + 3.4mL 1M NaH2PO4
300mM NaCl 60mL 5M
10% glycerol 100mL
400mM imidazole 27.23g
Lysis Buffer 500mL
20mM Na-PO4 8.96mL Na2PO4 + 1.04mL NaH2PO4 pH 7.8
20mM NaCl 2mL 5M
1mg/mL leupeptin
2mg/mL aprotinin pH 7.8
1M Na-PO4 Bufferà932mL 1M Na2PO4 + 68mL 1M NaH2PO4

For insect cells:
Lyse and dounce cells in lysis buffer 1mL/150mm plate
Bring Na-PO4 to 50mM and NaCl to 300mM
Add 560mL per 10mL(5M NaCl) Add 300mL 1M pH 7.8 Na-PO4
Dounce cells and spin at 45K for 1h.
Load over the column.
Collect fractions containing the desired protein from chelating column.
Dilute 6 fold with Buffer C (0M NaCl) + 50mM NaCl