Western Blot of TBP from TBP-GST Bacteria

Culture Bacteria

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

2. Shake at 37ºC ON.

Induce Bacteria

3. Adjust culture with LB until ON culture OD [at 600 nm] is ~0.6–0.8.

4. Induce with 40 mM IPTG (made fresh) at 1:100, shake for another 1–2 h.

5. Transfer to centrifuge tube and pellet bacteria (10 k rpm/10 min/RT).

6. Make up lysis solution (beta-mercaptoethanol (1:10) in 2x LSB- usually make up 1 ml total solution) and boil in 100°C bead bath for 10 min.

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

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

9. Store in –20°C.

Western Blot

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

11. Block in TBST w/ 5% milk ON at 4ºC.

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

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

14. ECL.