Western Blot of TBP from TBP-GST bacteria

Culture bacteria

1. Grow glycerol stock A13 (TBP-GST in BL21) in 10ml LB and 10μl  ampicillin (1:1000)

2. Shake @ 37℃ ON

Induce bacteria

3. Adjust culture with LB until ON culture OD [@ 600nm is ~0.6-0.8

4. Induce with 40mM IPTG (made fresh) @ 1:100, shake for another 1-2 hours5. Transfer to centrifuge tube and pellet bacteria (10k rpm/10 minutes/RT)

6. Make up lysis solution (beta-mercaptoethanol (1:10) in 2x LSB- usually make up 1mL total solution) and boil in 100℃ bead bath for 10 minutes

7. Add equal volume of lysis solution to pellet and boil for 10 minutes

8. Check to see if solution is uniform, if still goopy, add more lysis solution and reboil (total volume ~750μl )

9. Store in -20℃

Western Blot

10. Run out 1μl  of bacterial lysate in 9uL 2xLSB on gel (load all 10uL in one lane)

11. Block in TBST w/ 5% milk ON @ 4℃

12. Primary Ab- use TBP (58C9) 1:400 in TBST w/ 5% milk for 2 hours

13. Secondary Ab- use mHRP 1:25000 in TBST w/ 5% milk for 2 hours

14. ECL