In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay

Materials and Reagents

1. 1.5 mL reaction tubes (Sarstedt, catalog number: 72.690.001)

2. 100 mm square Petri dishes (Thermo Scientific, catalog number: 11349273)

3. HEPES (Fisher Bioreagents, catalog number: BP310-1)

4. NaCl (Fisher Chemicals, catalog number: S/3161/65)

5. MgCl₂ (Sigma, catalog number: 63064)

6. DTT (Melford Laboratories, catalog number: D11000)

7. ATP (Sigma-Aldrich, catalog number: A2383)

8. Ubiquitin (R&D Systems, catalog number: U-100H-10M)

9. NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, mini protein gels (Invitrogen, catalog number: NP0322BOX)

10. NuPAGE MES-SDS running buffer (20×) (Invitrogen, catalog number: NP0002)

11. NuPAGE LDS sample buffer (4×) (Invitrogen, catalog number: NP0007)

12. β-Mercaptoethanol (PanReac AppliChem, catalog number: A1108)

13. SeeBlue Plus2 pre-stained protein standard (Invitrogen, catalog number: LC5925)

14. Pierce^TM 20× TBS buffer (ThermoFisher, catalog number: 28358)

15. Tween-20 (Sigma, catalog number: P1379)

16. iBlot 2 nitrocellulose transfer stacks (Invitrogen, catalog number: IB23001)

17. Ethanol (VWR chemicals, catalog number: MFCD00003568)

18. Bovine serum albumin (BSA) (Sigma, catalog number: A3294)

19. Milk powder (Marvel)

20. Mono and polyubiquitylated conjugates, mAb (FK2) antibody (Ubiquigent, catalog number: 68-0121-500)

21. Ub-K63 mouse anti-human, clone: HWA4C4 (Affymetrix eBioscience^TM, catalog number: 14-6077-82)

22. HRP-conjugated anti-mouse antibody (Promega, catalog number: W4021)

23. Clarity Western ECL substrate (Bio-Rad, catalog number: 1705060)

24. Clarity Max Western ECL substrate (Bio-Rad, catalog number: 1705062)

25. Purified recombinant E1 (UBA1a, prepared under reducing conditions, storage: -70 °C) (Burge et al., 2020)

26. Purified recombinant E2 (UBC2, prepared under reducing conditions, storage: -70 °C) (Burge et al., 2020)

27. Purified recombinant E2 variant (UEV1, storage: -70 °C) (Burge et al., 2020)

28. Purified recombinant E3 (GST-BIRC2 or GST-RNF8, storage: -70 °C) (Ubiquigent, catalog number: 63-0015-025 or 63-0021-025)

29. di-ubiquitin, K63-linked (storage: -70 °C) (Ubiquigent, catalog number: 60-0107-010)

30. Reaction buffer (10×) (see Recipes)

31. SDS-PAGE sample buffer (4×) (see Recipes)

32. 1× TBSt (see Recipes)

Equipment

1. Dry block heater (Grant QBD1)

2. Protein electrophoresis equipment (XCell SureLock Mini-Cell, EI0001)

3. Protein transfer system (iBlot 2 Dry Blotting System, Thermo Fisher Scientific)

4. Gel rocker

5. Microplate shaker (Grant, PMS-1000i)

6. Chemi-blot imager (ChemiDoc Imaging System, Bio-Rad)

Procedure

1. Di-ubiquitin formation assay

   1. Prepare three reaction mixes in 1.5 mL tubes on ice containing the following reagents: 100 nM E1, 2.5 µM E2, 2.5 µM E2 variant, and 100 µM ubiquitin, in a buffer of 50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 2 mM DTT, and 5 mM ATP in a total volume of 40 µL. Typical reagent concentrations and volumes are given in Table 1. If a controlled reaction start is desired, the E1 or ATP can be omitted from the reaction mix during setup and added to initiate the reaction (mix by gently flicking the tube or stirring with a pipette tip during the final reagent addition). Three control reactions should also be performed, omitting either the E2, the E2 variant, or both the E2 and E2 variant, demonstrating that di-ubiquitin formation is not a consequence of activity from either the E2 or E2 variant alone, or from activity of the E1.

      
      

      Table 1. Reaction composition

      Component	Working concentration	Volume per reaction	Final concentration
      E1	4 µM	1 µL	100 nM
      E2	25 µM	4 µL	2.5 µM
      E2 variant*	25 µM	4 µL	2.5 µM
      E3**	5 µM	8 µL	1.0 µM
      Ubiquitin	1167 µM	3.4 µL	100 µM
      Reaction buffer	500 mM HEPES pH 7.5 1 M NaCl 100 mM MgCl2 20 mM DTT	4 µL	50 mM HEPES pH 7.5 100 mM NaCl 10 mM MgCl2 2 mM DTT
      ATP	100 mM	2 µL	5 mM
      dH2O	Up to 40 µL	Up to 40 µL	-

      *Can be excluded in the E3 cooperation assay

      **Include only in the E3 cooperation assay

      
      

   2. Incubate the reaction mixes in a dry block heater at 37 °C for 0 min, 30 min, and 90 min.

   3. Stop the reactions by adding 1× SDS-PAGE sample buffer (13.3 µL) (see Recipes).

   4. Heat the samples at 70 °C for 5 min using a dry block heater.

   5. Run 20 µL of each sample on a NuPAGE mini protein gel in 1× NuPAGE MES-SDS running buffer using an electrophoresis system. Load 5 µL of SeeBlue Plus2 pre-stained protein standard to a single lane of the gel to enable molecular weight comparison of bands detected later during the western blot. Run the gel at 200 V until the lowest molecular weight marker is approximately 0.25 cm from the bottom of the gel (approximately 35 min). If analysis of both ubiquitin conjugates and K63-linked ubiquitin conjugates is desired, run two gels with 20 µL samples from each reaction on each gel. When assessing the presence of K63-linked ubiquitin, a sample of 100 ng K63-linked di-ubiquitin should be included on the gel, which will act as a positive control when blotting with the Ub-K63 antibody.

   6. Incubate the gel in 20% ethanol in a square Petri dish for 10 min on a gel rocker at room temperature.

   7. Transfer the proteins to a nitrocellulose membrane using the iBlot 2 Dry Blotting System and an iBlot 2 nitrocellulose transfer stack according to the manufacturer’s instructions. Perform the transfer at 20 V for 1 min, then 23 V for 4 min, and finally 25 V for 7 min.

      
      

   Assessing the formation of ubiquitin conjugates using the FK2-antibody (Ubiquigent)

   8. Block the membrane by covering it with 5% BSA dissolved in 1× TBSt (see Recipes) in a square Petri dish on a microplate shaker at 4 °C overnight.

   9. Apply the mono and polyubiquitylated conjugates, mAb (FK2) antibody at a 1:1,000 dilution in 10 mL of 1% BSA in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker for 1 h.

   10. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker.

   11. Apply the HRP-conjugated anti-mouse secondary antibody at 1:10,000 dilution in 10 mL of 5% BSA in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker for 1 h.

   12. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker operating at speed 4.

   13. Apply clarity Western ECL substrate according to manufacturer’s instructions.

   14. Measure chemiluminescence using a suitable imaging system (e.g., ChemiDoc imaging system, Bio-Rad).

       
       

   Assessing the formation of K63-linked ubiquitin conjugates using the Ub-K63 antibody (ThermoFisher)

   15. Block the membrane by covering it with 5% (w/v) milk dissolved in 1× TBSt. Incubate in a square Petri dish on a microplate shaker at 4 °C for 1 h.

   16. Apply the Ub-K63 mouse antibody at 1:250 dilution in 5 ml of 5% milk in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker overnight.

   17. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker.

   18. Apply the HRP-conjugated anti-mouse secondary antibody at 1:10,000 dilution in 10 mL of 5% (w/v) milk in 1× TBSt in a square Petri dish. Incubate at 4 °C on a microplate shaker overnight.

   19. Wash the membrane in 1× TBSt for 10 min three times at room temperature in a square Petri dish on a microplate shaker.

   20. Apply clarity Western ECL substrate according to manufacturer’s instructions.

   21. Measure chemiluminescence using a suitable imaging system.

       
       

2. E3 cooperation assay

   1. Prepare reaction mixes in 1.5 mL reaction tubes on ice containing the following reagents: 100 nM E1, 2.5 µM E2, 1 µM E3, and 100 µM ubiquitin in a buffer of 50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 2 mM DTT, and 5 mM ATP in a total volume of 40 µL. Where relevant, 2.5 µM of an E2 variant can also be included (e.g., when involved in di-ubiquitin formation in conjunction with an E2). Typical reagent concentrations and volumes are given in Table 1. If a controlled reaction start is desired, the E1 or ATP can be omitted from the reaction mix during setup and added to initiate the reaction (mix by gently flicking the tube or stirring with a pipette tip during the final reagent addition).

      To determine whether the observed activity is due to the E3, four control reactions should be carried out, omitting either the E3, the E2, the E2 variant, or both the E2 and E2 variant. This allows for the assessment of any activity that may be a consequence of the E1 and E2s in the absence of the E3, and also whether the E3 works in cooperation with the E2 or E2 variant, alone or in combination.

   2. Incubate reactions in a dry block heater at 30 °C for 60 min or other times as required.

   3. Carry out steps A3–A21.

Data analysis

The FK2 antibody detects all ubiquitin conjugates, which are observed as chemiluminescent bands on the western blot. Comparison of these bands with the pre-stained ladder allows confirmation of di-ubiquitin formation. This is seen in Figure 2A, where incubation of Leishmania UBC2 and UEV1 for 30 or 90 min resulted in the formation of a clear band for di-ubiquitin (minor bands were also observed for UBA1a-Ub and UBC2-Ub complexes because these also contain a conjugated ubiquitin). The FK2 antibody can also be used to detect substrate and/or poly-ubiquitination, as desired in the E3 cooperation assay (Figure 3A). In this example, multiple ubiquitin conjugate bands were seen when Leishmania UBC2 was incubated with the human E3s BIRC2 or RNF8.

   Blotting with the Ub-K63 antibody allows the specific assessment of the formation of K63-linked ubiquitin conjugates (Figure 2B and Figure 3B). Thus, assessment of the same reaction products using both the FK2 and Ub-K63 antibodies enables the analysis of whether conjugates detected by the FK2 antibody are K63-linked. For example, in Figure 2, the di-ubiquitin band that was observed when blotting with the FK2 antibody (Figure 2A) was also observed when blotting with the Ub-K63 antibody (Figure 2B), indicating that the di-ubiquitin formed by UBC2 and UEV1 is K63-linked. However, in the E3 cooperation assay (Figure 3), conjugates produced by incubation of UBC2 with BIRC2 or RNF8 that were detected by the FK2 antibody (Figure 3A) were not detected by the Ub-K63 antibody (Figure 3B), suggesting that these conjugates are not K63-linked.

Figure 2. Di-ubiquitin formation assay. A) Blot using FK2 ubiquitin conjugate antibody. B) Blot using K63-linked ubiquitin antibody. Reproduced from Burge et al. (2020).

Figure 3. E3 Cooperation assay. A) Blot using FK2 ubiquitin conjugate antibody. B) Blot using K63-linked ubiquitin antibody. Reproduced from Burge et al. (2020).

Notes

1. UBA1a and UBC2 need to be purified under reducing conditions to maintain catalytic activity: 5 mM β-mercaptoethanol was included in the purification buffers for nickel affinity purification and 2 mM DTT or 1 mM TCEP for size exclusion chromatography. Proteins were stored at -70 °C in buffers containing 2 mM DTT or 1 mM TCEP (Note: UBA1a and UBC2 purified in buffers without reducing agents were not catalytically active). Avoid freeze-thaw cycles to increase the storage lifetime of the proteins.

2. Successful assays were achieved with the following changes in gel-running and transfer conditions: firstly, running samples on 4%–20% Mini-Protean TGX gels (Bio-Rad) in a running buffer of 25 mM Tris and 192 mM glycine pH 8.3, at 200 V for 35 min; secondly, incubating the gel, a nitrocellulose membrane, and filter paper in a transfer buffer of 25 mM Tris, 192 mM glycine, and 10% v/v methanol for 30 min before transfer using a Trans-Blot SD semi-dry transfer cell (Bio-Rad) at 25 V for 25 min.

3. If low activity is seen in the E3 cooperation assay, try increasing the incubation temperature to 37 °C.

Recipes

1. Reaction buffer (10×)

   500 mM HEPES pH 7.5

   1 M NaCl

   100 mM MgCl₂

   20 mM DTT

2. SDS-PAGE sample buffer (4×)

   95% NuPAGE LDS sample buffer (4×)

   5% β-mercaptoethanol

3. 1× TBSt

   5% (v/v) Pierce^TM 20× TBS buffer

   0.05% (v/v) Tween-20

   94.95% (v/v) deionized H₂O

Acknowledgments

This protocol was adapted from the research published in Burge et al. (2020). We would like to thank Ubiquigent for providing a 3 month internship for RB, during which this protocol was developed. This work was supported by a Medical Research Council Studentship to RB (MRC MR/N018230/1) and the Wellcome Trust to JCM (200807/Z/16/Z).

Competing interests

The authors declare no competing interests

References

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