酵母细胞裂解液泛素化蛋白检测方法

                  酵母细胞裂解液泛素化蛋白检测方法

                  Detection of a ubiquitinated protein from a yeast lysate

1. Grow cells O/N at 26°C (shaking).

2. Next day, cut back cells to .2 ODU/ml in 20 ml medium and grow at 26°C until they reach .5-1.0 ODU/ml.

3. Spin cells out for 5 min at room temp. Aspirate medium (do not decant).

4. Resuspend cells in 1 ml 5mM N-Ethylmaleimide (NEM) and transfer to an eppi tube. Prepare NEM immediately before use (7 mg NEM/ 10 ml H20).

5. Add 100 µl 100% TCA, vortex. Ice for 30 min.

6. Spin @ full speed for 10 min at 4°C, aspirate. Keep tubes on ice throughout the protocol. 7. Add 1 ml ice-cold acetone. Sonicate (using water bath sonicator) pellets into solution.

8. Repeat Steps 6 and 7.

9. Spin @ full speed for 10 min at 4°C, aspirate.

10. Dry pellets in speed vac.

11. Add 100 µl Urea Lysis Buffer + 5 mM NEM (fresh). Sonicate into solution.

12. Add 100 µl glass beads and vortex for 10 min.

13. Heat @ 75°C for 5 min, vortex 10 min.

14. Heat @ 75°C for 5 min.

15. Add 1 ml Tween IP buffer + 5 mM NEM (fresh).

16. Mix well by Inversion.

17. Spin full speed for 15 min at 4°C.

18. Transfer 950 µl of the sup to a new eppi tube. Careful not to disturb the pellet.

19. Add 5 µl of antibody against desired ubiquitinated protein.

20. Rotate O/N at 4°C.

21. Next day, add protein A sepharose beads to lysates.

22. Rotate for 2 hrs at 4°C.

23. Spin out beads full speed for 30 sec at 4°C. Carefully aspirate sup, leaving ~100µl sup so as not to disturb beads.

24. Wash beads 1x with Tween Urea buffer, 2x with Tween IP buffer and 1x with TBS.

25. Carefully remove the final 100µl of sup.

26. Dry beads in speed vac.

27. Add 50 µl sample buffer, tap beads into solution.

28. Boil samples at 75°C for 10 minutes.

29. Load 20 µl onto gel.

30. Run gel and transfer to nitrocellulose as usual.

31. See autoclaved blot protocol.

Urea Lysis Buffer

6 M Urea

1% SDS

50 mM Tris pH 7.5

1 mM EDTA

Tween IP Buffer 

50 mM Tris pH 7.5

150 mM NaCl

0.5% Tween-20

0.1 mM EDTA

Tween Urea Buffer 

100 mM Tris pH 7.5

200 mM NaCl

2 M Urea

0.5% Tween-20

               Autoclaving nitrocellulose membranes

                 to improve detection of ubiquitin immunoreactivity

1. Perform SDS-PAGE and transfer proteins to nitrocellulose according to your favorite protocol. (Do not use PVDF)

2. Rinse blot in distilled water for ~10 seconds. 3MM filter paper nitrocellulose membrane

3. Sandwich the blot between two sheets of filter paper and submerge as shown below. submerged in dH2O

4. Place in autoclave tray. Autoclave 30 min. liquid cycle.

5. Pour off excess water. The blot should remained sandwiched as in Figure 1 even though it is no longer submerged. Return sandwiched blot to autoclave. Autoclave 15 min. dry cycle. Figure 1. Sandwiching blot for autoclaving

6. Remove blot from from autoclave. Carefully disassemble blot after it has cooled. Use forceps and be careful not to tear the blot as you remove it from the filter paper.

7. Immunoblot with your favorite protocol, but see tips on the next page. I use pipet box lids as trays ~200 g some sort of weight: 100 ml bottle of water, etc.

Some additional tips...

- I have observed that milk (and pure casein) is not the very best blocking agent for use with horseradish peroxidase and chemilluminescence. For instance, blocking with 20% heat-inactived bovine calf serum gave a four-fold higher signal than blocking with milk in otherwise identical experiments.

- My current block is Tris-buffered saline + 0.45% Tween 20 + 20% heat-inactivated (65°C for 30 min.) bovine calf serum.

- Many of the commercial antibodies against ubiquitin work very poorly in yeast. The anti-ubiquitin mAb from Zymed (http://www.zymed.com) works well