Bacterial Expression of IRS-1 containing GST-fusion Proteins
1. Grow cells in 5ml (or more) of LB-Amp overnight for a starter culture.
2. Grow larger culture using the overnight culture as a seeding culture. The culture is grown in LB-Amp medium. Aerate well with shaking at 37 C until OD at 600nm is ~0.6.
3. Add the appropriate amount of IPTG stock solution to the culture to obtain a final IPTG concentration of ~2mM.
4. Continue shaking for several hours up to overnight.
5. Spin down cells and resuspend pellet in 1/100 initial volume of culture into lysis buffer -- PBS-CMF, 2mM EDTA, 10mM DTT, PMSF, benzamidine and 0.5M NaCl.
6. Cells are lysed by sonication at 4 C.
7. Add Triton X-100 to 1% final concentration.
8. Centrifuge at 20 - 30 K rpm in ultracentrifuge at 4 C for 30 min.
9. Filter sample through a 0.45m pore size membrane. Mix this filtered supernatant with glutathione Sepharose 4B which has been washed with PBS-CMF, 10mM DTT, 0.5M NaCl.
10. Rotate the filtered supernatant/sepharose at 4 C for 30-60min.
11. Spin, remove liquid and wash the sepharose 2x with PBS-CMF, 10mMDTT, 0.5M NaCl.
12. Elute the fusion protein from the sepharose 3x with 10mM glutathione, 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.
13. Dialyze fusion protein against 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.