Ubiquitination assay

Ubiquitination assay

Concerning the feasibility in the experiments, we provide two methods for detection of the ubiquitination of the protein of interest (POI). To ensure the ubiquitination detected was only from the POI, but not its interacting proteins, the ubiquitination assay is performed under denaturing conditions of either 2% SDS or 6 M guanidine.

Ubiquitination assay under 2% SDS denaturing condition

1. Transfect cells with plasmids expressing tagged POI (e.g., HA) and ubiquitin (e.g., FLAG) for protein overexpression for 48 hrs.

2. Before harvest, rinse cells with cold PBS for 3 times.

3. Lyse cells using RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholic acid, 1% NP-40) containing 2% SDS, 10 mM deubiquitinases inhibitor NEM (N-Ethylmaleimide), protease and phosphatase inhibitors.

4. Boil at 95-100 °C for 10 mins.

5. Sonicate until the solution is clear.

6. Centrifugate lysates at 16000 × g, 4°C for 15 mins, collect the supernatants.

7. Input sample preparation. Collect 1/10 of total supernatants. Add 1x Laemmli sample buffer containing 10% 2-Mercaptoethanol to the supernatant and heat samples at 95-100°C for 10 mins as Input sample.

8. Dilute the supernatants with 10 times RIPA lysis buffer to reduce the SDS concentration to 0.2%.

9. Incubate the supernatants with anti-HA antibody under rotation overnight at 4°C.

10. Next day, Pre-wash the Protein A/G PLUS-Agarose beads with cold PBS twice and cold RIPA lysis buffer once.

11. Add the pre-washed Protein A/G PLUS-Agarose beads to the supernatants for further incubation at 4°C for 2 h.

12. Wash POI-antibody bound beads four times in RIPA lysis buffer containing 0.1% SDS.

13. After the last wash, add 1x Laemmli sample buffer containing 10% 2-Mercaptoethanol to the beads and heat samples at 95-100°C for 10 mins as IP sample.

14. Run SDS-PAGE to separate the immunoprecipitated POI and co-immunoprecipitated ubiquitin for immunoblotting.

Ubiquitination assay under 6 M guanidine denaturing condition

1. Transfect cells with plasmids expressing tagged POI (e.g., HA) and His-ubiquitin for protein overexpression for 48 hrs.

2. Before harvest, rinse cells with cold PBS for 3 times.

3. Detach cells using 0.25 Trypsin-EDTA.

4. Input sample preparation. Lyse 1/10 of total cells using RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholic acid, 1% NP-40) containing protease and phosphatase inhibitors. Add 1x Laemmli sample buffer containing 10% 2-Mercaptoethanol to the supernatant and heat samples at 95-100°C for 10 mins as Input sample.

5. Lyse the other 9/10 of total cells using Buffer A (6 M guanidine-HCl, 0.1 M Na₂HPO₄/NaH₂PO₄, 10 mM imidazole [pH 8.0]) containing 10 mM NEM, protease and phosphatase inhibitors.

6. Sonicate until the solution is clear.

7. Centrifugate lysates at 16000 × g, 4°C for 15 mins, collect the supernatants.

8. Pre-wash the nickel-nitrilotriacetic acid (Ni-NTA) beads (QIAGEN) with Buffer A for 3 times.

9. Incubate the supernatants with the pre-washed Ni-NTA beads under rotation for 3 h at room temperature.

10. Wash the beads once in Buffer A.

11. Wash the beads twice in Buffer B (a mixture of Buffer A: Buffer TI at a ratio of 1:3).

12. Wash the beads once in Buffer TI (25 mM Tris-HCl, 20 mM imidazole [pH 6.8]).

13. After the last wash, add 1x Laemmli sample buffer containing 10% 2-Mercaptoethanol to the beads and heat samples at 95-100°C for 10 mins as Pulldown sample.

14. Run SDS-PAGE to separate the immunoprecipitated His-ubiquitin and pulled-down POI for immunoblotting.