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Star Republic: Guide for Biologists

Quick screen of recombinant protein expression

Edited by Chang Zhu
1. Inoculate 2 ml LB/antibiotic media with a single colony for each sample.

2. Grow cells overnight at 37 C with shaking (225-250 rpm).

3. Add 100 ml of the overnight culture to 5 ml LB/antibiotic media.

4. Grow cells for 2-4 hours or until the cells are in mid-log phase (OD600 = 0.5).

5. Remove 1 ml aliquot of cells to a 1.5 ml eppendorf tube, centrifuge at 12,000 rpm in a mcirocentrifuge for 30 seconds, remove media, and freeze the cell pellet at -20 C.

6. Add IPTG to the final concentration of 1 mM, continue incubation for 2-4 hours at 37 C with shaking.

7. Remove 1 ml aliquot of cells to a 1.5 ml eppendorf tube, centrifuge at 12,000 rpm in a mcirocentrifuge for 30 seconds, remove media, and freeze the cell pellet at -20 C. (Optional: take samples at several time points.)

8. Resuspend cells in 200 ml of 1X Tris-glycine SDS-Polyacrylamide Gel loading buffer.

9. Boil the tubes for 5 minutes, then centrifuge at maximum speed for 5 minutes.

10. Analyze 5-20 ml on a SDS-PAGE gel. Run samples from the same colony (before and after IGTP induction) side-by-side.

11. After completion of SDS-PAGE gel, staining the gel. Colony expressing the recombinant protein should show extra bands in the induced samples.