Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays

Materials and Reagents

1. HA-ubiquitin-vinyl sulfone (Boston Biochem, catalog number: U-212)

2. 26S proteasome (Ub-VS treated) (Ubiquigent, catalog number: 65-1020-010)

3. Recombinant purified His₆-T7-UBP6 (Skelly et al., 2019) or other DUB of interest

4. Ubiquitin subtrates for deubiquitination assays:

   Poly-ubiquitin (Ub3-7) K48-linked (Boston Biochem, catalog number: UC-220)

   Poly-ubiquitin (Ub3-7) K63-linked (Boston Biochem, catalog number: UC-320)

   Di-ubiquitin K48-linked (Boston Biochem, catalog number: UC-200B)

   Di-ubiquitin K63-linked (Boston Biochem, catalog number: UC-300B)

5. UltraPure Tris (Invitrogen, catalog number: 15504-020)

6. MgCl₂ hexahydrate (Sigma-Aldrich, catalog number: M2670)

7. Adenosine triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209)

8. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 1114740005)

9. Glycerol (Sigma-Aldrich, catalog number: G5516)

10. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771)

11. Bromophenol blue (Sigma-Aldrich, catalog number: B8026)

12. Anti-HA antibody (ThermoFisher, catalog number: 26183)

13. Anti-ubiquitin antibody (Santa Cruz Biotechnology, catalog number: sc-8017)

14. Antibody against the DUB being analysed, e.g., anti-T7 for His₆-T7-UBP6 (Millipore, catalog number: 69522)

15. Double-distilled H₂O (ddH₂O)

16. 15% SDS-PAGE gel (e.g., compatible with Bio-Rad Mini-PROTEAN system)

17. SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher, catalog number: 34577)

18. 10× DUB labelling buffer (store at -20°C) (see Recipes)

19. 10× deubiquitination buffer (store at -20°C) (see Recipes)

20. 2× SDS-PAGE loading buffer (see Recipes)

21. DUBs, proteasomes, HA-UbVS, and ubiquitin substrates (see Recipes)

Equipment

1. 1.5 ml microcentrifuge tubes (e.g., Starlab, catalog number: S1615-5500)

2. Pipettes and tips (e.g., Gilson Pipettman, various volumes)

3. Heat block (e.g., ThermoFisher, catalog number: 88870004)

4. Protein gel electrophoresis and western blotting apparatus (e.g., Bio-Rad Mini-PROTEAN system)

Procedure

1. HA-UbVS labelling

   1. Set up 10-μl reactions including the following: 1 μl 10× DUB labelling buffer, 5 μl 700 nM DUB, 1.25 μl 80 nM 26S proteasome (if required), 1 μl 7 μM HA-UbVS, and 1.75 μl ddH₂O. Control samples should be included in which components are omitted and replaced with ddH₂O.

   2. Incubate for 30 min at room temperature or in a heat block at 22°C.

   3. Add 10 μl 2× SDS-PAGE loading buffer to each sample.

   4. Incubate for 10 min in a heat block at 70°C.

   5. Perform SDS-PAGE with a 15% gel and western blotting using standard methods with:

      1. Anti-HA antibodies (at 1:5000 dilution) to detect free HA-UbVS (~9.8 kDa) and HA-UbVS-labelled DUB (Figure 1).

      2. Antibodies (at antibody-specific dilutions) against the appropriate DUB or epitope tag to detect both unlabelled and labelled DUB. Any standard imaging system can be used depending on the secondary antibodies (e.g., film or CCD imaging system for chemiluminescence).

         
         

      
      Figure 1. HA-UbVS labelling of recombinant purified His₆-T7-UBP6. A typical western blot showing free and HA-UbVS-labelled recombinant His₆-T7-UBP6. Adapted from Figure 6B of Skelly et al. (2019).

      
      

2. In vitro deubiquitination assays

   1. Set up 10 μl reactions including the following: 1 μl 10× deubiquitination buffer, 1 μl 200 nM DUB, 1 μl 12.5 nM 26S proteasome (if required), 1 μl 4 μM ubiquitin substrate, and 6 μl ddH₂O. Control samples should be included in which components are omitted and replaced with ddH₂O.

   2. Incubate in a heat block at 30°C for the desired time [2-16 h for His₆-T7-UBP6 (Skelly et al., 2019), but dependent on the DUB and substrate].

   3. Add 10 μl 2× SDS-PAGE loading buffer to each sample.

   4. Incubate for 10 min in a heat block at 70°C.

   5. Perform SDS-PAGE with a 15% gel and western blotting using standard methods with anti-ubiquitin antibodies to detect the original ubiquitin chain substrate and observe any DUB-mediated chain disassembly as lower-order ubiquitin species. Any standard imaging system can be used depending on the secondary antibodies (e.g., film or CCD imaging system for chemiluminescence).

Recipes

1. 10× DUB labelling buffer (store at -20°C)

   500 mM Tris pH 7.4

   50 mM MgCl₂

   10 mM DTT

   10 mM ATP

2. 10× deubiquitination buffer (store at -20°C)

   500 mM Tris pH 7.4

   50 mM MgCl₂

   10 mM DTT

   50 mM ATP

3. 2× SDS-PAGE loading buffer (store at room temperature, add DTT fresh before use)

   20% glycerol

   120 mM Tris pH 6.8

   4% SDS

   0.02% Bromophenol Blue

   100 mM DTT

4. DUBs, proteasomes, HA-UbVS, and ubiquitin substrates

   Diluted in ddH₂O from stocks to the required concentrations described above

Acknowledgments

This work was supported by a Royal Society University Research Fellowship (UF090321), a BBSRC grant (BB/L006219/1), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 678511). This protocol was derived from Skelly et al. (2019).

Competing interests

The authors declare that no competing interests exist.

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