This is a cached page for the URL (http://www.whitelabs.org/Lab%20Protocols/bacterial%20protocols/Bacterial%20Expression%20of%20IRS-1-containing%20fusion%20proteins.htm). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Bacterial Expression of IRS-1 containing GST-fusion Proteins

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.