GST融合蛋白純化方法 Purification of GST Fused Proteins
日期：2009-08-10 17:44:39
Materials and Reagents
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STE Buffer
10 mM Tris-HCl, pH 8.0
1 mM EDTA
150 mM NaCl -
Lysozyme solution
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PBS
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Elution Buffer
20 mM GSH -
10% Sarkosyl in STE Buffer
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10% Triton X-100 in STE Buffer
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1 M DTT
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100 mM IPTG
Procedure
Day 1
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Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.
Day 2
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Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicillin.
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Grow at 37oC to an A600 of 0.6 to 0.8.
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Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37oC or grow overnight at room temperature.
Lower IPTG concentrations and lower growing temperatures tend to produce greater solubility at the expense of yield. -
Pellet cells by centrifuging at 3000 g, 4oC for 10 min. Decant media and resuspend cells in 30 ml ice-cold PBS to wash. Transfer to a 40-ml Oak Ridge tube and centrifuge at 3000 g, 4oC for 10 min. Decant PBS.
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This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells.
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Thaw pellet on ice if cells are frozen else proceed to the next step.
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Resuspend pellet in 10 ml of ice cold STE Buffer.
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Add 100 ml of freshly prepared lyozyme solution, incubate on ice for 15 min. Just before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by inversion and sonicate for a total time of 1 min.
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Centrifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Transfer supernatant to a 50-ml conical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20 ml. The effective concentration of Sarkosyl and Triton X-100 will be 0.7% and 2% respectively. Incubate at room temperature for 30 min.
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Pour the lysate to 1 ml bed of prepared Glutathione Sepharose in PBS. Incubate at room temperature for 30 min to 1 hr with agitation.
To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, invert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and resuspend beads with 1 ml of PBS. -
Wash the beads with 3 X 50 ml of PBS. Finally resuspend in 5 ml of PBS. Pour to a dispo-column. Wash the 50-ml conical tube with an additional 5 ml of PBS. Pool with the first 5 ml in the dispo-column.
To wash, use the same centrifugation technique for preparing the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips. -
If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDS PAGE.
