Protocol for integrated ubiquitination analysis of in vitro E3 ligase-DUB regulation and in vivo ubiquitin chain linkage characterization

Summary

Here, we present a protocol for the recombinant production of DTX3L and USP28, along with in vitro and in vivo procedures for examining their regulatory functions. We describe steps for reconstituting DTX3L-dependent ubiquitination in vitro with purified UBA1, UbE2D1, ubiquitin, and DTX3L and assessing USP28-mediated deubiquitination. We then detail procedures for the in vivo detection of ubiquitin chain linkage-type through co-immunoprecipitation and Western blotting. This adaptable approach enables analysis of regulatory crosstalk between E3 ligases and deubiquitinases within the ubiquitin system.

For complete details on the use and execution of this protocol, please refer to Mennerich et al.¹

Graphical abstract

Highlights

• •Instructions for analyzing mechanistic crosstalk between E3 ligases and DUBs
• •Steps for recombinant production of E1, E2, E3, and DUB enzymes
• •Procedures for distinguishing ubiquitin chain linkages in vivo using coIP
• •Adaptable workflow applicable to diverse ubiquitination-deubiquitination enzyme pairs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for the recombinant production of DTX3L and USP28, along with in vitro and in vivo procedures for examining their regulatory functions. We describe steps for reconstituting DTX3L-dependent ubiquitination in vitro with purified UBA1, UbE2D1, ubiquitin, and DTX3L and assessing USP28-mediated deubiquitination. We then detail procedures for the in vivo detection of ubiquitin chain linkage-type through co-immunoprecipitation and western blotting. This adaptable approach enables analysis of regulatory crosstalk between E3 ligases and deubiquitinases within the ubiquitin system.

Before you begin

The identification of protein ubiquitination in vitro requires the recombinant expression of the components of the ubiquitination cascade. Here we describe a protocol using UBA1 (ubiquitin-activating enzyme), UbE2D1 (ubiquitin-conjugating enzyme, alternatively known as UbcH5A), DTX3L (a RING-type ubiquitin E3 ligase), ubiquitin (UniProt code: P0CG48), and USP28 (a counteracting deubiquitinating enzyme). Using DTX3L and USP28 as examples of an E3 ligase and its counteracting deubiquitinase, we outline methods for their recombinant expression and functional analysis in vitro.

While most proteins in this protocol are expressed efficiently in E. coli, full-length DTX3L (residues 1–740 of isoform 1; UniProt code: Q8TDB6-1) requires expression in the SF21 insect cell line using a baculoviral system due to its size and complexity. To facilitate bacterial expression and functional studies, a truncated but ubiquitin-ligase-competent variant of DTX3L comprising the RING-DTC domains (residues 555–740) can be cloned into a bacterial expression vector. This approach enables reconstitution of ubiquitination activity while simplifying protein production.

UBA1 and UbE2D1 are expressed in Rosetta2(DE3), USP28 in the MDS42 strain of E. coli, and ubiquitin in BL21(DE3). The reconstitution of the ubiquitination cascade described here can be adapted to other pairs of cognate E2 and E3 proteins with catalytic activity.

For in vivo experiments, HEK-293 cells, plasmid DNA, and respective detection reagents are required. The methods described here are specific for the detection of DTX3L (ubiquitin E3 ligase) and USP28 (deubiquitinating enzyme) ubiquitination in vivo. Ubiquitination of HA-tagged DTX3L and FlagM2-tagged-USP28 is detected by co-immunoprecipitation (CO-IP) using specific a-HA-Tag and a-FlagM2-Tag antibodies together with Protein G Sepharose™ 4 fast flow beads, followed by Western blot analysis. These protocols are also adaptable for detecting other ubiquitinated proteins of interest.

Innovation

This protocol presents key technical innovations in ubiquitination detection, including optimized reaction conditions for in vitro ubiquitination assays using linkage-specific ubiquitin chain inhibitors and real-time monitoring of ubiquitination dynamics. It further introduces a robust and highly reproducible workflow for in vivo ubiquitination analysis that enables clear discrimination among different polyubiquitin chain types. Together, these advances improve the reproducibility and operability of ubiquitination detection by offering a streamlined and dependable framework for connecting enzymatic ubiquitination activity to cellular ubiquitin signaling. The protocol also utilizes calcium-phosphate transfection in HEK-293 cells, ensuring strong expression of ubiquitination components while maintaining experimental consistency and cost-efficiency. Overall, this integrated approach provides enhanced mechanistic insights into ubiquitin-dependent regulatory processes.

Institutional permissions

The biosafety level required for HEK-293 cells varies between countries and regulatory jurisdictions. Therefore, researchers must verify the applicable biosafety classification based on their local institutional or national biosafety guidelines.

Recombinant protein expression in E. coli for the in vitro ubiquitination assay

Timing: 3 days

This section describes the protocol for recombinant protein expression in E. coli. Although the expression vectors used may differ, the protocol is applicable to all bacterial expression vectors mentioned here.

• 1.
  Transform chemicompetent E. coli strains, either BL21(DE3) or Rosetta2(DE3) with the expression plasmids encoding the proteins of interest, respectively.

  • a.
    Prepare LB-agar plates containing the appropriate antibiotic.
  • b.
    Thaw the chemicompetent E. coli cells on ice, 25 μL cells/per plasmid in separate reaction tubes.
  • c.
    Add 1 – 10 ng of each plasmid to each tube containing cells and gently mix.
  • d.
    Incubate the cells on ice for 30 min.
  • e.
    Place the tubes for 45 sec in a heat block or water bath warmed up to 42°C.
  • f.
    Move the cells back to ice and incubate for 5 min.
  • g.
    Add 1 mL of antibiotic-free SOC or LB media to the cells.
  • h.
    Incubate in a shaking incubator for 1 h at 37°C and 180 – 200 rpm.
  • i.
    Plate 100 – 200 μL of the cell suspension on the prepared LB-agar plates.

    Note: The LB-agar plates may be pre-warmed prior to plating of the cells to 37°C to increase transformation efficiency.

  • j.
    Incubate the plates for 14 – 18 h at 37°C.
• 2.
  Express the recombinant proteins.

  • a.
    For each expression plasmid use three to four colonies from the agar plate to inoculate 10 mL of LB-media supplemented with the corresponding antibiotics.
  • b.
    Incubate for 14 – 18 h at 37°C and 180 – 200 rpm in a shaking incubator.
  • c.
    For each expression construct, prepare 500 mL of sterile TB-autoinduction media (TBAIM) supplemented with adequate antibiotics.
  • d.
    Use 5 mL of the overnight culture to inoculate the TBAIM and grow each culture separately in a 5 L Erlenmeyer flask.
  • e.
    Incubate the cultures in a shaking incubator at 37°C and 140 – 200 rpm until the O.D. reaches 0.6–1.0. Depending on the cell line, this is achieved between 3 – 5 h.

    Note: The shaking speed depends on the rotation radius of the incubator. For wide-angle incubators, 140 – 160 rpm is enough, while narrow-angle incubators require higher rpm.

  • f.
    Decrease the temperature to 18°C and incubate for 20 h.
  • g.
    Collect the cell pellet by centrifuging the cultures at 4200×g for 30 min and discard the supernatant. Determine the weight of the cell pellets; for this, the weight of the empty tubes must have been determined beforehand.
  • h.
    Re-suspend the pellets in lysis buffer [50 mM HEPES (pH 7.5), 500 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol, 10 mM imidazole] using 25 mL of buffer per 10 g of pellet.

    Pause point: The resuspended pellets can be stored at −20°C or below until the proteins are purified.

Protein purification with affinity tags

Timing: 2 days per protein

• 3.
  Lyse the cells.

  • a.
    Thaw the cells from the previous step in a water bath at room temperature (RT) (20 – 25°C).
  • b.
    Add DNase and Pefabloc SC protein inhibitor to the cell suspension at final concentrations of 20 μg/mL and 0.1 M, respectively, and mix by inversion.
  • c.
    Transfer the cells to a vessel submerged in an ice slurry.
  • d.
    Sonicate for a total of 2.5 min of active sonication at a 40% amplitude and cycles of 5 s of sonication with idling periods of 15 s to prevent overheating.

    Note: The sonication time, both total and of active and idling periods, as well as the intensity may vary depending on the type of sonicator used.

  • e.
    Transfer the lysates to centrifuge compatible tubes.
  • f.
    Centrifuge the lysates for 30 – 45 min at 27.600×g at 4°C to recover soluble protein.
  • g.
    Carefully transfer the supernatant to a 50 mL syringe and filter using a 0.45 μm sterile syringe filter into a 50 mL tube in ice.
• 4.
  Purify the proteins by immobilized metal Ion affinity chromatography (IMAC).

  • a.
    Prepare a 5 mL HiTrap IMAC HP column by charging it with NiSO₄ and equilibrating it with 5 column volumes (CV) of lysis buffer.
  • b.
    Load the filtered lysate onto the column at a flowrate of 1 mL/min.
  • c.
    Wash the column with 5 – 10 CVs of lysis buffer.
  • d.
    Wash the column with 5 – 10 CVs of wash buffer [50 mM HEPES (pH 7.5), 500 mM NaCl, 10% (v/v) glycerol, 0.5mM TCEP, 25mM imidazole].

    Note: Due to the expression strain used, USP28 requires an additional wash to remove chaperone proteins. Use 5 CVs ATP-wash buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 10 mM imidazole, 0.5 mM TCEP, 5 mM ATP, 1 mM MgCl₂].

  • e.
    Elute the proteins with 3 – 5 CVs of elution buffer [50 mM HEPES (pH 7.5), 500 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP, 300 mM imidazole], in fractions of 0.5–2 mL.

    Note: At this point the IMAC purification fractions can be analyzed by SDS-PAGE to select the relevant fractions for further purification. Alternatively select elution fractions with concentrations above 1 mg/mL, estimated by the absorbance at 280 nm.

    Optional: For USP28 this can be a pause point. The pooled IMAC elution fractions can be stored at 4°C overnight after diluting with SEC buffer in a 1:10 ratio to reduce the imidazole concentration and prevent precipitation. Alternatively, proceed directly to step 8.

    Note: After this step, Ube2D1 is treated overnight with SUMO-specific protease 2 (SENP2) to cleave the His-sumo tag. Proceed directly to step 6.

    Note: After this step dialyze UBA1 against anion exchange (AnEX) low salt buffer [20 mM Tris (pH 8), 50 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol] up to 24 h at 4°C using a dialysis membrane with a 6 – 8 kDa molecular weight cutoff to reduce the concentration of NaCl.

• 5.
  Purification with MBP trap (only for DTX3L).

  • a.
    Equilibrate two 5 mL MBP trap columns with 5 – 10 CVs of gel filtration buffer [30 mM HEPES (pH 7.5), 350 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol].
  • b.
    Load the IMAC elution onto the column at a flow rate of 1mL/min.
  • c.
    Wash the columns with 3 – 5 CVs of gel filtration buffer.
  • d.
    Elute the protein with 3 – 5 CVs of MBP elution buffer [30 mM HEPES (pH 7.5), 350 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol, 10 mM maltose], collecting fractions of 0.5–2 mL.
  • e.
    Estimate the protein concentration by measuring absorbance at 280 nm and combine fractions with concentrations above 0.5 mg/mL.
• 6.
  Perform affinity purification tag cleavage and reverse IMAC (only done for Ube2D1 and DTX3L).

  • a.
    Mix the protein with the respective protease, SENP2 for Ube2D1 and TEV for DTX3L. The final molar concentration should be approximately 1/30 of the concentration of the uncleaved target protein.
  • b.
    Dialyze Ube2D1 against gel filtration buffer to remove imidazole for up to 24 h at 4°C using a dialysis membrane of 6 – 8 kDa molecular weight cutoff.

    Note: DTX3L does not need to be dialyzed as the imidazole was removed during the MBP trap purification step.

  • c.
    Prepare a 5 mL HiTrap IMAC HP column like in step 2.a.
  • d.
    Load the protein/protease mix onto the column at a flowrate of 1 mL/min and collect the flow-through containing the target protein.
  • e.
    Wash with lysis buffer until the target protein has eluted from the column.

    Note: As an alternative to washing only with lysis buffer, after 2 – 3 CVs of lysis buffer, the column could also be washed with 2 – 3 CVs of R.IMAC wash buffer [50 mM HEPES (pH 7.5), 500 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol, 50 mM imidazole] to ensure protein recovery.

• 7.
  Anion exchange chromatography (only to be done for UBA1).

  • a.
    Equilibrate a 5mL HiTrap Q XL column with 5 – 10 CVs of AnEX low salt buffer.
  • b.
    Load the dialyzed UBA1 onto the column at a flowrate of 1 mL/min.

    Note: To improve the binding efficiency, the flow-through can be re-circulated through the column once.

  • c.
    Wash the column with 5 CVs of AnEX low salt buffer.
  • d.
    Elute the contents of the column using a gradient AnEX low salt buffer to AnEX high salt buffer [20 mM Tris (pH 8), 500 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol] over 20 min at 2 mL/min.
  • e.
    Combine the fractions containing the protein and proceed with size exclusion.
• 8.
  Purify the proteins by size exclusion chromatography (SEC).

  • a.
    Equilibrate a preparative size exclusion column with at least 1.5 CVs of SEC buffer [30 mM HEPES (pH 7.5), 350 mM NaCl, 0.5 mM TCEP, 10% (v/v) glycerol].
  • b.
    Concentrate the protein solution obtained from their respective previous steps to approximately 1 mL using a 15 mL Amico Ultra centrifugal filter with a 3 or 30 kDa molecular weight cutoff depending on the theoretical size of the target protein.
  • c.
    Transfer the concentrated protein solution into a 1.5 mL reaction tube and centrifuge for 10 min at >16.000×g and 4°C to remove possible debris or protein precipitates.
  • d.
    Recover the supernatant and load it onto the equilibrated column, collect fractions.
  • e.
    Analyse the SEC fractions by SDS-PAGE. Pool and concentrate those with the highest purity with a 15 mL Amicon ultra centrifugal filter to a concentration of 5 – 20 mg/mL.
  • f.
    Aliquot the protein in PCR reaction tubes in volumes of 10 – 50 μL per tube and flash freeze with liquid nitrogen.
  • g.
    Store at −70 °C or below.

Pause point: The purified proteins can be stored in aliquots until their use. We have not observed any change in activity, even two years after purification.

Purification of ubiquitin

Timing: 2 days

• 9.Lyse the cells as in step 3 from the previous section but omit the filtering step of the supernatant after centrifugation.
• 10.
  Precipitate the protein with trichloroacetic acid:

  • a.
    Without filtering, carefully recover the soluble fraction of the lysate in a 50 mL falcon tube and allow to equilibrate at RT (20–25°C) for up to 30 min.
  • b.
    Incubate the supernatant at 80°C using a water bath for 5 min.
  • c.
    Centrifuge the boiled suspension for 30 min at 30.000×g to remove protein aggregates.
  • d.
    Carefully decant the soluble fraction into a second 50 mL falcon tube.
  • e.
    Add trichloroacetic acid (TCA) to the supernatant to a final concentration of 5% (v/v) and incubate for 10 min at RT (20 –25°C).
  • f.
    Centrifuge for 2 min at 3000×g and 4°C to pellet the proteins.
  • g.
    Aspirate the supernatant with a pipette and re-suspend the pellet in 15 mL of phosphate buffer saline (PBS).

    Note: The solution can be turbid after this step.

  • h.
    Adjust the pH of the solution to 7.4 with 1 M NaOH.
  • i.
    Centrifuge the protein solution for 5 min at 3000×g and 4°C to remove the proteins that do not go back into solution.
  • j.
    Recover the supernatant and dialyse it overnight (up to 24 h) against cation exchange (CatEX) low salt buffer [50 mM Ammonium acetate (pH 4.5)].
• 11.
  Cation exchange chromatography.

  • a.
    Prepare a 5 mL HiTrap SP FF column and equilibrate it with 5 – 10 CVs of CatEX low salt buffer.
  • b.
    Load the dialysed protein solution onto the column at a flowrate of 1 mL/min.

    Note: To improve binding efficiency, the flow-through can be re-circulated through the column once.

  • c.
    Wash the column with 5 CVs of CatEX low salt buffer.
  • d.
    Elute the contents of the column using a gradient from CatEX low salt buffer to CatEX high salt buffer [50 mM Ammonium acetate (pH 4.5), 500 mM NaCl] over 20 min at 2 mL/min collecting fractions.
  • e.
    Analyse the fractions by SDS-PAGE and combine those that contain the target protein.
  • f.
    Concentrate with a 15 mL Amicon Ultra centrifugal filter with a 3 kDa molecular weight cutoff to up approximately 1 mL.
  • g.
    Purify the protein with SEC as in step 8 from the previous subsection.

    Pause point: The purified protein can be stored in aliquots until their use. We have not observed any change in activity, even two years after purification.

Cell preparation for the in vivo ubiquitination assay

Timing: 1 week

• 12.
  Thaw the frozen HEK-293 cells.

  • a.
    Remove the cryovial containing frozen HEK-293 cells from liquid nitrogen tank and thaw the cells by gentle agitation in a 37°C water bath. The vial should thaw quickly – within about 1 to 2 min.

    Note: To reduce the possibility of contamination, keep the O-ring and the cap out of the water and as soon as the content is thawed, decontaminate the vial by using 70% ethanol.

    CRITICAL: When handling liquid nitrogen, always use proper protective equipment, such as cryo-gloves and face shield. Work in a well-ventilated area to prevent oxygen displacement.

  • b.
    Transfer the vial content to a 15 mL falcon tube containing 8 mL of pre-warmed complete growth medium (MEM supplemented with 10% fetal bovine serum, 1x MEM non-essential amino acids, 100 U/mL penicillin and 100 mg/mL streptomycin).
  • c.
    Centrifuge the cell suspension at approximately 200×g for 5 min.
  • d.
    After centrifugation, discard the supernatant carefully by aspiration, resuspend the cell pellet in 1-2 mL pre-warmed fresh complete MEM and dispense the cells into a 75 cm² cell culture flask containing 10 mL of pre-warmed fresh complete MEM.
  • e.
    Incubate cells in a humidified atmosphere containing 5% CO₂ at 37°C.
• 13.
  Sub-culture of HEK-293 cells.

  • a.
    Passage HEK-293 cells when they reach 80%–90% confluency.
  • b.
    Aspirate carefully the cell culture media and wash the cells once by adding ∼8 mL of Dulbecco’s phosphate-buffered saline (DPBS).
  • c.
    Remove carefully the DPBS by aspiration and add 1.5 mL of 1x Trypsin-EDTA solution (0.5% Trypsin-EDTA) to the 75 cm² cell culture flask. Ensure that the Trypsin-EDTA solution is equally distributed and completely covers the monolayer cells.
  • d.
    Incubate cells in a humidified atmosphere with 5% CO₂ at 37°C for 1 to 2 min.

    Note: To prevent cells from clumping, avoid hitting or shaking the cell culture flask while waiting for the cells to detach. Ensure that the cells are detached from the cell culture flask by using the microscope.

    Note: The amount of Trypsin-EDTA solution depends on the size of the cell culture flask or cell culture dish used. Furthermore, the amount of time needed to dissociate cells using Trypsin-EDTA solution varies according to the cell type.

  • e.
    When the cells are detached from the cell culture flask add 5 mL pre-warmed complete fresh growth medium (MEM) to inhibit tryptic activity.
  • f.
    Transfer the cell suspension to a 15 mL falcon tube and centrifuge at approximately 200×g for 5 min.
  • g.
    Aspirate the supernatant carefully while avoiding contact with the cell pellet.
  • h.
    Resuspend the cell pellet in 2 mL fresh cell culture medium and transfer the cells either to a 75 cm² cell culture flask containing 10 mL of pre-warmed complete cell culture medium (1:10 split ratio) for sub-culture or seed the cells into a 10 cm cell culture dish containing 8 mL of pre-warmed complete cell culture medium (split ratio 1:5) for experiments.
  • i.
    Incubate cells in a humidified atmosphere with 5% CO₂ at 37°C.

    CRITICAL: All steps should be carried out under strict aseptic conditions.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
USP28 (Dilution: 1:1000)	Sigma-Aldrich	#HPA006778; RRID:AB_1080520
Ubiquitin (Dilution: 1:1000)	BioLegend	#646304; RRID:AB_2734516
HA-Tag (F-7) (Dilution: 1:1000)	Santa Cruz	#sc7392; RRID:AB_2894930
Anti-Flag® M2 (Dilution: 1:5000)	Sigma-Aldrich	#F1804; RRID:AB_262044
α-Tubulin (Dilution: 1:10.000)	Sigma-Aldrich	#T5168; RRID:AB_477579
Goat anti-mouse IgG HRP (Dilution: 1:5000)	Bio-Rad	#1706516; RRID: N/A
Goat anti-rabbit IgG HRP (Dilution: 1:5000)	Bio-Rad	#1706515; RRID: N/A
Bacterial and virus strains
E. coli BL21 (DE3)	New England BioLabs	#C2527
E. coli Rosetta2 (DE3)	Sigma-Aldrich	#71397
Sf21 insect cells	Expression systems	#94-003 S
Chemicals, peptides, and recombinant proteins
TB Auto-induction media	Formedium	# AIMTB0260
Insect-XPRESS™ Protein-free Insect Cell Medium	Lonza	# 12-730Q
ATP (Adenosine 5′-triphosphate disodium salt hydrate)	Sigma-Aldrich	# A2383
TEV protease	Sigma-Aldrich	# T4455
SENP2 protease	MedChemExpress	# HY-P71288
Pefabloc SC	Roche	# 11429868001
DNase I	Roche	# 10104159001
Critical commercial assays
MycoAlert Mycoplasma Detection Kit	Lonza	#LT07-118
Pierce™ ECL Western Blotting Substrate	Thermo Scientific™	#32209
Experimental models: Cell lines
HEK 293	ATCC	#CRL-1573; RRID:CVCL_0045
Recombinant DNA
pMJS162-USP28	Mennerich et al.1	N/A
DTX3L deletion mutants in pcDNA3.1 HA	Mennerich et al.1	N/A
pDZ-Flag-USP28	Flügel et al.2	N/A
pcDNA3.1-DTX3L-HA	Mennerich et al.1	N/A
pRK5-HA-Ubiquitin-WT (Addgene)	Kah et al.3	#17608
pRK5-HA-Ubiquitin-KO (Addgene)	Kah et al.3	#17603
pRK5-HA-Ubiquitin-K48 (Addgene)	Kah et al.3	#17605
pRK5-HA-Ubiquitin-K63 (Addgene)	Kah et al.3	#17606
Expression vector for UBA1 (Addgene)	Berndsen et al.4	#34965
Expression vector for UbE2D1 (Addgene)	Wiener et al.5	#61081
Expression vector for full-length DTX3L	Ashok et al.6	N/A
Expression vector for ubiquitin (Addgene)	Brzovic et al.7	#12647
Other
Branson 450 Digital Sonifier with ½-inch stepped horn.	Branson	Discontinued
Minisart sterile syringe filters (PES, 0.45μm, 28mm)	Sartorius	#16533-K
Spectra/Por Dialysis membrane (6–8 kDa)	Fisher Scientific	#11435849
5 mL HiTrap IMAC HP column	Merck	#GE17-0920-05
5 mL MBPTrap HP column	Merck	#GE28-9187-79
5 mL HiTrap Q XL column	Merck	#GE17-5159-01
5 mL HiTrap SP FF column	Merck	#GE17-5157-01
Superdex 75 pg 16/600 HiLoad preparative Size exclusion chromatography column	Merck	#GE28-9893-33
Superdex 200 pg 16/600 HiLoad preparative Size exclusion chromatography column	Merck	#GE28-9893-35
Amicon Ultra 15 mL centrifugal filter (3 kDa)	Merck	#UFC900324
Amicon Ultra 15 mL centrifugal filter (30 kDa)	Merck	#UFC903024
Amicon Ultra 15 mL centrifugal filter (100 kDa)	Merck	#UFC910024
cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail Tablets	Merck	#11836170001
Protein G Sepharose™ 4 Fast Flow	Merck	#GE17-0618-01
Amersham™ Protran® Western blotting membranes, nitrocellulose (0.45μm)	Merck	#GE10600002

Materials and equipment

Plasmid information for recombinant protein production

Protein (amino acid boundaries)	UniProt ID	Vector backbone (expression system; strain)	Affinity tags for purification purposes
UBA1 (1 – 1058)	P22314	pET21d (E. coli; Rosetta2(DE3))	Non-cleavable N-terminal 6×His tag
UbE2D1 (1 – 147)	P51668	pETSUMO (E. coli; Rosetta2(DE3))	SENP2-cleavable N-terminal 6×His tag
DTX3L (1 – 740)	Q8TDB6	PFastBac1 His-MBP (insect cells; SF21)	TEV protease –cleavable N-terminal 6×His MBP tags
Ubiquitin (1 – 76)	P0CG48	PET15 (E. coli; BL21(DE3))	Untagged
USP28 (1 – 1077)	Q96RU2	PMJS162 (E. coli; MDS42)	Non-cleavable N-terminal 6×His tag

Buffers for protein purification

Lysis buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
2 M imidazole (pH 7.5)	10 mM	5 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

IMAC wash buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
2 M imidazole (pH 7.5)	25 mM	12.5 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

ATP wash buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	150 mM	30 mL
Glycerol	10% (v/v)	100 mL
2 M imidazole (pH 7.5)	10 mM	5 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
0.1 M ATP	2 mM	20 mL
1 M MgCl2	5 mM	5 mL
Total	N/A	1000 mL

Note: The buffer can be aliquoted in 50 mL tubes and stored at −20 °C for up to six months. Otherwise, the buffer needs to be freshly prepared.

IMAC elution buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
2 M imidazole (pH 7.5)	300 mM	150 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

MBP elution buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	30 mM	60 mL
5 M NaCl	350 mM	70 mL
Glycerol	10% (v/v)	100 mL
1 M maltose	10 mM	10 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

Reverse IMAC buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
2 M imidazole (pH 7.5)	50 mM	25 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

AnEX low salt buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M Tris (pH 8)	20 mM	40 mL
5 M NaCl	50 mM	10 mL
Glycerol	10% (v/v)	100 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

AnEX high salt buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M Tris (pH 8)	20 mM	40 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

AnEX high salt buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M Tris (pH 8)	20 mM	40 mL
5 M NaCl	500 mM	100 mL
Glycerol	10% (v/v)	100 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

CatEx low salt buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M Ammonium acetate (pH 4.5)	50 mM	100 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

CatEx high salt buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M Ammonium acetate (pH 4.5)	50 mM	100 mL
5 M NaCl	500 mM	100 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

SEC buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	30 mM	60 mL
5 M NaCl	350 mM	70 mL
Glycerol	10% (v/v)	100 mL
0.5 M TCEP (pH 7.5)	0.5 mM	1 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

Ubiquitination buffer

Reagent	Final concentration	Amount (for 1000 mL)
0.5 M HEPES (pH 7.5)	50 mM	100 mL
5 M NaCl	50 mM	10 mL
Total	N/A	1000 mL

Note: Store at 4°C for up to six months.

20× ubiquitin activation buffer

Reagent	Final concentration	Amount (for 5 mL)
5 M Tris (pH 7.5)	1 M	1 mL
0.5 M DTT (pH 7.5)	40 mM	0.4 mL
0.1 M ATP	40 mM	2 mL
1 M MgCl2	100 mM	0.5 mL
Total	N/A	5 mL

Note: The buffer can be aliquoted as 100 μL aliquots in PCR tubes, flash frozen with liquid nitrogen and store at −20°C or below for up to a year.

Reagents for HEK-293 cell transfection with calcium-phosphate technique

2x HEPES buffer (pH 7.05)

Reagent	Final concentration	Amount (for 100 mL)
1 M HEPES pH 7.05	50 mM	5 mL
5 M NaCl	280 mM	5.6 mL
1 M Na2HPO4	1.5 mM	150 μL
ddH2O	N/A	Up to 100 mL
Total	2x	100 mL

Adjust to pH 7.05 with NaOH. Sterilize the buffer by filtration through a 0.45 μm filter.

Note: Store at −20°C in 10 mL aliquots.

Calcium Chloride 2.5 M

Reagent	Final concentration	Amount (for 50 mL)
CaCl2 ∗ 2H2O (dihydrate)	2.5 M	18.03 g
ddH2O	N/A	Up to 50 mL
Total	2.5 M	50 mL

Note: Store at −20°C in small aliquots.

Reagents for total protein isolation from transfected HEK-293 cells and co-immunoprecipitation

Cell lysis buffer for co-immunoprecipitation (CO-IP)

Reagent	Final concentration	Amount (for 10 mL)
10x TBS	1x	1 mL
10% Triton-X100	1%	1 mL
0.5 M EDTA pH 8	2 mM	40 μL
0.5 M EGTA pH 8	2 mM	40 μL
125 mM Na4P2O7	10 mM	800 μL
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Note: Prepare buffer always fresh and add one protease inhibitor tablet (1 per 10 mL buffer) before use.

Note: Unused cell lysis buffer can be frozen in small aliquots (1 – 2 mL) at −20°C and thawed once. This is cost-effective and sustainable.

Washing buffer for CO-IP

Reagent	Final concentration	Amount (for 50 mL)
10x TBS	1x	5 mL
10% Triton-X100	1%	5 mL
0.5 M EDTA pH 8	2 mM	200 μL
0.5 M EGTA pH 8	2 mM	200 μL
125 mM Na4P2O7	10 mM	4 mL
ddH2O	N/A	Up to 50 mL
Total	N/A	50 mL

Note: Store at 4°C for up to 1 week.

Bradford Reagent

Reagent	Final concentration	Amount (for 1000 mL)
SERVA Blue G	0.1%	100 mg
Ethanol (95%)	4.75 %	50 mL
Phosphoric acid (85 %)	8.5 %	100 mL
ddH2O	N/A	Up to 1000 mL
Total	N/A	1000 mL

Note: Store in a brown glass bottle to avoid photodegradation at RT (20-25°C).

Alternatives: Ready-to-use Bradford Protein Assay Kits are also available, e.g. Thermo Scientific™ (#23200).

Reagents for Western blot analysis

2x SDS-PAGE loading buffer

Reagent	Final concentration	Amount (for 10 mL)
1 M Tris/HCl pH 6.8	125 mM	1.25 mL
SDS	4%	0.4 g
Glycerol (99%)	20%	2.02 mL
Bromophenol Blue	0.01%	1 mg
ddH2O	N/A	Up to 10 mL
Total	N/A	10 mL

Note: Add 80 μL 0.5 M DTT per 1 mL buffer (final concentration 40 mM) before use.

Note: Store buffer (w/o DTT) in small aliquot at −20°C.

CRITICAL: Sodium dodecyl sulfate (SDS) powder has been demonstrated to pose a significant hazard when inhaled due to its capacity to function as a highly effective surfactant. This property of SDS powder leads to irritation and subsequent damage of respiratory tissues. To enhance safety and precision, the utilization of SDS pellets is strongly advised, as they significantly reduce dust formation and are widely regarded as a more secure option.

Alternatives: Ready to use 2× and 4× SDS-PAGE loading buffers are also available, e.g. BioRad 2× Laemmli Sample Buffer (#161-0737) and 4× Laemmli Sample Buffer (#161-0747).

SDS-PAGE separating gel (7.5%), gel thickness 1 mm

Reagent	Final concentration	Amount (for 9 mL)
30% Acrylamide/Bis-solution (37.5:1)	7.5%	2.25 mL
1.875 M Tris/HCl pH 8.8	400 mM	1.92 mL
10% SDS	0.1%	0.09 mL
10% APS	0.1%	0.09 mL
TEMED	0.1%	0.01 mL
ddH2O	N/A	Up to 9 mL
Total	N/A	9 mL

Note: Prepare the solution immediately before use.

SDS-PAGE stacking gel (5%), gel thickness 1 mm

Reagent	Final concentration	Amount (for 4 mL)
30% Acrylamide/Bis-solution (37.5:1)	5%	0.667 mL
0.6 M Tris/HCl pH 6.8	60 mM	0.4 mL
10% SDS	0,1%	0.04 mL
10% APS	0.1%	0.04 mL
TEMED	0.1%	0.0025 mL
ddH2O	N/A	Up to 4 mL
Total	N/A	4 mL

Note: Prepare the solution immediately before use.

Note: Volumes given are sufficient for small (8.5 x 10 x 1 mm) gel format.

CRITICAL: Prepare solutions always under a fume hood. Unpolymerized acrylamide is highly toxic, readily absorbed through the skin, and classified as a neurotoxin and suspected carcinogen, requiring strict use of gloves, and eye protection. Once polymerized into a gel acrylamide is far less hazardous, but it must still be disposed of as chemical waste. Further, TEMED is toxic and corrosive, can irritate the skin, eyes, and respiratory tract, and must always be used with appropriate personal protective equipment.

Alternatives: Pre-cast gels are also available, e.g. BioRad (#4561023).

10x SDS-PAGE running buffer

Reagent	Final concentration	Amount (for 1000 mL)
Tris base	250 mM	30 g
Glycine	1920 mM	144 g
SDS	0.1%	10 g
ddH2O	N/A	Up to 1000 mL
Total	N/A	1000 mL

Note: Store at RT (20-25°C).

Anode buffer 1

Reagent	Final concentration	Amount (for 1000 mL)
Tris base	300 mM	36.4 g
Methanol	20%	200 mL
ddH2O	N/A	Up to 1000 mL
Total	N/A	1000 mL

Adjust to pH 11.3 with 1 M boric acid.

CRITICAL: Adjust pH before adding methanol.

Note: Buffer can be stored at RT (20–25°C).

Anode buffer 2

Reagent	Final concentration	Amount (for 1000 mL)
Tris base	25 mM	6 g
Methanol	20%	200 mL
ddH2O	N/A	Up to 1000 mL
Total	N/A	1000 mL

Adjust to pH 10.6 with 1 M boric acid.

CRITICAL: Adjust pH before adding methanol.

Note: Buffer can be stored at RT (20–25°C).

Cathode buffer

Reagent	Final concentration	Amount (for 1000 mL)
Tris base	25 mM	6 g
Methanol	20%	200 mL
ddH2O	N/A	Up to 1000 mL
Total	N/A	1000 mL

Adjust to pH 9 with 1 M boric acid.

CRITICAL: Adjust pH before adding methanol.

Note: Buffer can be stored at RT (20–25°C).

Ponceau S Solution

Reagent	Final concentration	Amount (for 500 mL)
Ponceau S	0.25%	1.25 g
Glacial acetic acid	15%	75 mL
Methanol	40%	200 mL
ddH2O	N/A	Up to 500 mL
Total	N/A	500 mL

Note: Solution can be re-used and stored at RT (20–25°C) for up to 1 year.

CRITICAL: Methanol containing buffers are highly flammable and toxic even when diluted. All work should be done in a fume hood with proper personal protective equipment, including nitrile gloves and eye protection. Methanol containers must be kept tightly closed and away from heat or sparks. In addition, methanol vapors and skin absorption pose significant health risks, so minimizing exposure and ensuring proper waste disposal are essential for safe use.

Step-by-step method details

In vitro ubiquitination assay to investigate DUB and E3 ligase activity

Timing: 2 days

The following protocol uses UBA1 as the ubiquitin activating enzyme, UbE2D1 as the ubiquitin conjugating enzyme, DTX3L as the ubiquitin E3 ligase capable of automodification and USP28 as a deubiquitinase.

• 1.
  Auto ubiquitination of DTX3L.

  • a.
    Prepare the ubiquitination reaction in a micro tube on ice as follows:

    Reagent (stock concentration)	Final concentration	Volume
    UBA1 (74.80 μM)	0.6 μM	0.4 μL
    UbE2D1 (121.31 μM)	2 μM	0.8 μL
    DTX3L (48.47 μM)	1 μM	1 μL
    Ubiquitin (1576.36 μM)	45 μM	1.4 μL
    USP28 (22.76 μM)	5 μM	11 μL
    Ubiquitin activation buffer (20×)	1×	2.5 μL
    Ubiquitination buffer	N/A	32.8 μL

    Note: Add the ubiquitin activation buffer last, as it will start the reaction.

  • b.
    Incubate the mix at RT (20 – 25°C) for four hours.

    Note: Adding USP28 or the DUB of interest at the beginning of the incubation period will digest poly-ubiquitin as it forms. To evaluate poly-ubiquitin hydrolysis after a specific time, start the reaction without USP28, then add it at the desired time point.

    Note: To study ubiquitination of a confirmed or putative substrate, add the substrate protein at the beginning of the reaction. While optimal substrate concentration may require optimization, we recommend 5 μM as a starting concentration.

  • c.
    Add 12.5 μL of 4× SDS loading buffer to stop the reaction.
  • d.
    Mix thoroughly and incubate for 5 min in a heat block or water bath at 95°C.
  • e.
    Spin the sample for 1 min at a maximum speed of 15000×g.
  • f.
    Resolve the sample with a 4 – 20% SDS PAGE gel.

    Note: Although fixed concentration gels, e.g. 7.5 or 10%, can be used, gradient gel allow a more homogeneous separation of proteins across a wide high and low molecular weight range.

  • g.
    Analyze the ubiquitination reaction by Western Blot using an anti-ubiquitin antibody.
  • h.
    Typical ubiquitination blots are shown in Figure 1.

Figure 1.

In vitro hydrolysis of pre-formed poly-ubiquitin chains

(A) To examine the hydrolysis of pre-formed poly-ubiquitin chains, reactions were incubated at 30, 60 or 120 min, as indicated at the top of the figure panel, before adding USP28. After the addition of USP28, the reactions were incubated for 2 h and subsequently stopped by adding Laemmli sample buffer. The reactions were analyzed by Western blot analysis with a specific antibody against ubiquitin.

(B and C) The hydrolysis was assessed under both time-dependent and enzyme-dependent conditions by adding varying concentrations of USP28 to the reactions. The increasing concentration of USP28 is represented by an increase in size of orange dots. Hydrolysis was stopped by adding Laemmli sample buffer. The reactions were analyzed by Western blotting with specific antibodies against ubiquitin (A–C) or USP28 (B).

Data are reproduced from Mennerich et al.¹

In vivo ubiquitination assay in HEK-293 cells via calcium-phosphate transfection followed by co-immunoprecipitation

Timing: 2 days

Seeding of HEK-293 cells into 10 cm cell culture dishes for co-immunoprecipitation and plasmid DNA transfection using calcium-phosphate technique.

• 2.
  Seed cells one day (∼24 h) prior to transfection.

  • a.
    Follow the “before you begin” step 2 to seed the cells.
  • b.
    Seed the cells into 10 cm cell culture dishes with ∼60% confluency for transfection.
• 3.
  Transfect cells with plasmids using calcium-phosphate technique the next morning.

  • a.
    Check cell morphology and density under a microscope. Cells should be at 70-80% confluency for transfection. Change the medium 2 h prior to transfection. Use a volume of 6 mL complete growth media for a 10 cm cell culture dish.
  • b.
    Transfect the cells with indicated plasmids with calcium-phosphate technique.

    Tube no.	Plasmid DNA
    	Flag-USP28 5 μg	HA-DTX3L 5 μg	Ubiquitin-WT 2.5 μg	Ubiquitin-KR 2.5 μg	Ubiquitin-K48 2.5 μg	Ubiquitin-K63 2.5 μg
    1	X	X	–	–	–	–
    2	X	X	X	–	–	–
    3	X	X	–	X	–	–
    4	X	X	–	–	X	–
    5	X	X	–	–	–	X
    –	Transfection-Mix 2× HEPES: CaCl2 2.5 M: Flag-USP28 HA-DTX3L Plasmid-DNA (Ubiquitin) ddH2O Total transfection volume:		Amount 300 μL 30 μL × μL × μL × μL Up to 600 μL 600 μL		Final concentration 1× 125 mM 5 μg 5 μg 2.5 μg N/A N/A

  • c.
    Prepare the tubes by adding the calculated amount of ddH₂O.
  • d.
    Add plasmid DNA first, then add CaCl₂.
  • e.
    Add 2x HEPES dropwise under vortexing and incubate the mixture 5-10 min at RT (20-25°C) until it gets cloudy.

    Note: It is crucial to prepare the transfection mix in polystyrene tubes to prevent the DNA from adhering to the tube.

    Note: To clearly observe the DNA-calcium-phosphate precipitate, the polystyrene tube must be transparent.

    CRITICAL: If cloudiness is absent after preparation of the transfection mix, verify the pH of the 2x HEPES buffer. An accurate adjustment of the pH is critical for proper DNA precipitate formation and successful transfection.

  • f.
    Add the mixture dropwise to the cells and mix carefully by shaking the cell culture dish.
  • g.
    Incubate cells in a humidified atmosphere with 5% CO₂ at 37°C for 24 h.
  • h.
    4 h before harvesting the cells change the media (6 mL pre-warmed complete growth medium) and add MG 132 to a final concentration of 50 μM.
  • i.
    Harvest the cells for co-immunoprecipitation.

Co-immunoprecipitation for the detection of ubiquitinated proteins by Western blot

Timing: 3 days

Isolation of total proteins from transfected HEK-293 cells, protein quantification using Bradford assay, equilibration of Protein G Sepharose™ 4 Fast Flow beads, and co-immunoprecipitation followed by the detection of ubiquitinated HA-DTX3L and FlagM2-USP28 detected by Western blot with specific antibodies.

• 4.
  Total protein isolation from transfected HEK-293 cells.

  • a.
    Discard the medium from the cell culture dishes.
  • b.
    Wash cells once with 1x PBS.

    Note: If excessive cell loss occurs during the washing step, we recommend omitting the wash. Instead, aspirate as much medium as possible by holding the plate at an angle and carefully removing the medium from the wall of the plate to minimize cell detachment.

  • c.
    Add 300 μL lysis buffer for CO-IP.
  • d.
    Scape the cells and transfer the cell suspension into a 1.5 mL Eppendorf (EP) tube.
  • e.
    Put the EP tube on a lab roller at 4°C for 20 min.
  • f.
    Centrifuge the cell suspension at 4°C for 15 min.
  • g.
    Transfer the supernatant into a new 1.5 mL EP tube.

    Note: All steps should be performed on ice.

    Note: After total protein isolation, the EP tube must be kept on ice during all following steps.

• 5.
  Bradford assay.

  • a.
    Prepare a series of BSA standard solutions of known concentrations (e.g., 2, 4, 6, 8, 10, 12 μg/μL) by diluting a stock solution in ddH₂O in a total volume of 100 μL.
  • b.
    Dilute the protein sample by using 98 μL ddH₂O + 2 μL of the supernatant.
  • c.
    Use 100 μL ddH₂O as blank.
  • d.
    Add 900 μL of Bradford reagent and incubate the samples at RT (20–25°C) for 5 – 10 min.
  • e.
    Measure the absorbance at 595 nm using a spectrophotometer.
  • f.
    Calculate the protein concentration of the samples by comparing the absorbance values to the standard curve.

CRITICAL: Do not freeze the cell lysates before co-immunoprecipitation.

• 6.
  Equilibration of Protein G Sepharose™ 4 Fast Flow beads.

  • a.
    Add 300 μL beads into a 2 mL EP tube (∼30 μL beads per sample).

    Note: Ensure the beads are well-suspended before use by vortexing. Slightly cut off the pipette tip at the end before handling the beads.

  • b.
    Add 1.5 mL of washing buffer.
  • c.
    Vortex the EP tube and centrifuge at 1000×g for 4 – 5 min at RT (20-25°C).
  • d.
    Remove the supernatant carefully with a pipette without touching the beads.
  • e.
    Repeat step 5b – 5d; in total 3 times.
  • f.
    After the last washing step, resuspend the Sepharose beads in 1.2 mL of cell lysis buffer for CO-IP (110 μl per sample) and store them at 4°C or on ice until use.

    Note: Equilibrated Protein G Sepharose™ 4 Fast Flow beads can be stored at 4°C for up to 1 week.

    Pipetting scheme for CO-IP

    Sample no.	Sample concentration	a-FlagM2	a-HA tag	Protein G Sepharose™ 4 fast flow beads	Cell lysis buffer for CO-IP
    1	150 μg	2 μg	/	110 μL	Up to 390 μL
    2	150 μg	2 μg	/	110 μL	Up to 390 μL
    3	150 μg	2 μg	/	110 μL	Up to 390 μL
    4	150 μg	2 μg	/	110 μL	Up to 390 μL
    5	150 μg	2 μg	/	110 μL	Up to 390 μL
    1	150 μg	/	2 μg	110 μL	Up to 390 μL
    2	150 μg	/	2 μg	110 μL	Up to 390 μL
    3	150 μg	/	2 μg	110 μL	Up to 390 μL
    4	150 μg	/	2 μg	110 μL	Up to 390 μL
    5	150 μg	/	2 μg	110 μL	Up to 390 μL

• 7.
  Co-immunoprecipitation of ubiquitinated proteins with specific antibodies.

  • a.
    Dilute 150 μg of the protein sample in cell lysis buffer for CO-IP in a total volume of 390 μL.
  • b.
    Add 2 μg of either a-FlagM2 antibody for the precipitation of ubiquitinated USP28 or 2 μg of a-HA-Tag antibody for the precipitation of ubiquitinated DTX3L.
  • c.
    Incubate the samples on a lab roller at 4°C for 1 – 2 h.
  • d.
    Add 110 μL of equilibrated Protein G Sepharose™ 4 Fast Flow beads.

    Note: Vortex the equilibrated beads before use and slightly cut off the pipette tip at the end before pipetting the beads.

  • e.
    Incubate the samples on a lab roller at 4°C for 16 h.
  • f.
    After incubation, centrifuge at 400×g at 4°C for 5 min.
  • g.
    Remove the supernatant carefully without touching the beads.
  • h.
    Add 1 mL of washing buffer to the beads, vortex and centrifuge at 400×g at 4°C for 5 min.
  • i.
    Repeat step 6f – 6h; in total 5 times.
  • j.
    Resuspend the beads in 30 μL of 2× SDS loading buffer and boil the samples at 96°C for 6 min.

    Pause point: After boiling, samples can be stored at −20°C until SDS-PAGE.

• 8.
  Preparation of whole cell lysate (Input) samples for Western blot.

  • a.
    Prepare 20 – 50 μg from the same samples used for CO-IP.
  • b.
    Add 2x SDS loading buffer to get a final concentration of 1x.
  • c.
    Boil the samples at 96°C for 6 min.

Pause point: After boiling, samples can be stored at −20°C until SDS-PAGE.

• 9.
  SDS-Page gel preparation.

  • a.
    Prepare a 7.5% separation gel. Follow the instruction given under Material and Equipment. Cast the gel, leaving approximately 1 – 1,5 cm below the bottom of the comb out for the concentrating gel.
  • b.
    Remove the bubbles at the top of the gel with a layer of 70% EtOH.
  • c.
    Let the gel completely polymerize.

    Note: Polymerization should not be longer than 20 – 30 min.

  • d.
    Remove 70% EtOH and wash once with water.
  • e.
    Prepare a 5% stacking gel. Follow the instruction given under Material and Equipment. Cast the gel on top of the separation gel and add the comb (11 – 13 wells).
  • f.
    Let the gel completely polymerize.
  • g.
    Remove the comb.
  • h.
    Fill the electrophoresis chamber with 1x SDS-PAGE running buffer and place the gel into the chamber.
  • i.
    Load whole CO-IP samples (∼30 μL with ubiquitinated proteins), input (20 – 50 μg) and a molecular weight protein ladder into the wells. Separate the proteins by electrophoresis.

    Note: Using a 13-well comb allows the marker to be loaded between the CO-IP samples and whole cell lysate (Input) samples. After protein transfer, the membrane can be cut and incubated separately with the required antibodies. This approach is more sustainable and reduces reagent consumption.

• 10.
  Electrophoresis and protein transfer by semi-dry Western blot.

  • a.
    Run the gel(s) at 50 V for approximately 30 min until the samples reach the separating gel. Increase to 100 V for approximately 1 h. Stop immediately when the blue front reaches the bottom of the gel.
  • b.
    After electrophoresis, remove the gel from the running chamber and equilibrate it by soaking in cathode buffer for 15 min.
  • c.
    Equilibrate the Nitrocellulose (NC) membrane in anode buffer 2 for 15 min.
  • d.
    Assemble the transfer apparatus according to instructions in Figure 2.

    Note: Make sure that air bubbles are removed.

  • e.
    Transfer proteins from the gel to the NC membrane by applying 1.2 mA/cm² (∼59 mA/membrane) for 1 h 20 min.
  • f.
    After protein transfer, stain the membrane with Ponceau S for ∼1 min.
  • g.
    Destain first with ddH₂O until the protein marker bands appear.
  • h.
    Destain the NC membrane completely with 1x PBS + 0.1% Tween-20.

    Note: If a pre-stained protein marker was used, steps 9f - 9h are not necessary.

    • i.
      Block the membrane with 5% non-fat dry milk in 1x PBS + 0.1% Tween-20 for 1 h at RT (20-25°C).
  • j.
    Incubate the membrane with Direct-Blot™ HRP anti-Ubiquitin antibody (Dilution 1:1000 in 5% non-fat dry milk in 1x PBS + 0.1% Tween-20) overnight at 4°C with gentle agitation.

    Note: Antibody can be re-used for 2 – 3 times. Store at −20°C.

  • k.
    After a-ubiquitin antibody incubation, wash the NC membrane 3× in 1× PBS + 0.1% Tween-20 for 15 min.
  • l.
    Use ECL system for chemiluminescent detection of ubiquitination. Typical ubiquitination blots are shown in Figure 3: (A) USP28 and (B) DTX3L.

Figure 2.

Western blot transfer apparatus

Schematic representation of a semi-dry transfer apparatus setup for protein blotting. Layers are arranged from cathode (top) to anode (bottom). A 1 kg weight is applied on top to ensure uniform contact between layers during transfer.

Figure 3.

In vivo ubiquitination assay

HEK-293 cells were co-transfected with HA-DTX3L and Flag-USP28 expression vectors together with either an empty vector (Ctl), wild-type ubiquitin (Ub), a lysine-null mutant (Ub-KR), or mutants that retained only K48 (Ub-K48) or K63 (Ub-K63) and analyzed by Western blotting. (A) Ubiquitinated USP28 was detected after FlagM2-mediated and (B) ubiquitinated DTX3L was detected after HA-mediated immunoprecipitation with an ubiquitin antibody. Data are reproduced from Mennerich et al.¹

Expected outcomes

In vitro ubiquitination assay

The poly-ubiquitination of a given protein is recognized by a smear that appears above the molecular weight of the unmodified protein. This process can be visualized in SDS-PAGE with appropriate staining. To enhance the reliability of the results, the ubiquitination can be detected with an anti-ubiquitin antibody (Figure 1). The detection can be further explored with antibodies for the different proteins in the reaction if desired. Furthermore, the reaction can be modified to accommodate different ligases or deubiquitinases or even scaled up to use the ubiquitinated protein in downstream studies.

In vivo ubiquitination assay

To assess which ubiquitin linkages DTX3L uses to modify USP28, we performed ubiquitination assays with wildtype ubiquitin (Ub) and a lysine-null mutant (UbKR). As expected, USP28 ubiquitination was detectable with wildtype (Ub) but not with UbKR. In addition, we could also specify which ubiquitin linkage type contributes to chain formation. We found that DTX3L can form both K48- and K63-linked chains on USP28 using single lysine ubiquitin mutants (UbK48 and UbK63) (Figure 3A).

To determine which linkage USP28 removes from DTX3L, we repeated the assays with wild-type ubiquitin and the same mutants. As expected, DTX3L ubiquitination was detectable with the wild-type construct, while the UB-KR mutant abolished DTX3L ubiquitination. Furthermore, we observed that both K48- and K63-linked chains can be added to DTX3L, but USP28 selectively removes K63-linked ubiquitin, as shown by reduced K63-linked smearing (Figure 3B).

Limitations

The in vitro ubiquitination protocol is a straightforward method for testing the autoubiquitination of a ubiquitin E3 ligase or a substrate. As with many in vitro assays, the production of recombinant proteins is a limiting step. In the context of reconstituting the ubiquitination cascade, this is crucial to emphasize the need to purify at least four proteins. The majority of these proteins require specialized instrumentation to ensure the effective purification of the target proteins. This protocol involves the expression of DTX3L using a baculoviral expression system in SF21 insect cells. This system is considered time-consuming for laboratories that do not use it routinely. However, a viable alternative is to express only the domain with E3 ligase activity in E. coli, a method that can be employed to circumvent the aforementioned challenges.

The in vivo ubiquitination protocol has been optimized specifically for HEK-293 cells, and while the described cost-efficient calcium-phosphate transfection technique works efficiently in this cell line, it may not be directly transferable to other cell types without additional optimization. The workflow is designed for the study of overexpressed proteins in HEK-293 cells, with a focus on HA-DTX3L and FlagM2-USP28, though it can also be applied to other tagged proteins of interest. When adapting the protocol to examine endogenous protein levels, the primary limitation is antibody performance. Therefore, each antibody must be carefully validated to ensure specificity and suitability under experimental conditions.

Troubleshooting

Problem 1

Lack or weak poly-ubiquitin smear is detected in the in vitro assay.

Potential solution

It is important to keep store all proteins appropriately (−80°C in multiple aliquots of small volumes: 10 to 50 μL) and avoid freeze-thaw cycles that could negatively affect the activity of the enzymes.

If the assay is done with ligases or fragments therefrom for which no studies in vitro have been previously done, the compatibility between E2 and E3 should be evaluated from literature review a priori. Alternatively, a selection of E2 ubiquitin conjugating enzymes can be tried in the assay as the E2/E3 combination can affect the outcome of the reaction.

While the ubiquitin activating buffer with ATP does not necessarily need to be prepared fresh and can be stored at −80°C for at least six months after flash freezing in liquid nitrogen, we have noticed that old buffer (stored for over a year) results in overall lower ubiquitination activity. For new assays, we recommend the buffer to be prepared fresh.

Problem 2

Detection with anti-ubiquitin antibody results in a smear, but the visualization after probing with antibodies specific to the protein of interest does not show a smear or shows a single band.

Potential solution

Some E2s like Ube2N have been shown to produce free poly-ubiquitin (Soss et al.), the same applies to certain E3 ligases like UBE3A/E6AP (Ronchi et al.). If the smear detected with the anti-ubiquitin antibody does not reflect with other antibodies, it might be possible that while the E3 ligase does not undergo auto-modification, it can form free ubiquitin chains in the absence of a substrate. This would require further verification, like the addition of a known or putative substrate that could act as a ubiquitin acceptor.

Problem 3

Lack or weak ubiquitin smear detectable in vivo after Western blot.

Potential solution

Transfection efficiency may be too low, resulting in insufficient expression of HA-DTX3L or FlagM2-USP28. Transfect HEK-293 cells with HA-DTX3L or FlagM2-USP28 individually, perform a standard Western blot and detect overexpressed proteins using a-HA Tag or a-FlagM2 antibodies. Strong bands at the expected molecular weights indicate successful transfection. In the absence of bands, optimize DNA quality and verify that the pH of the 2x HEPES buffer is exactly adjusted to 7.05, as this is essential for efficient calcium-phosphate transfection technique.

The DNA concentration of the different overexpressed ubiquitin plasmids may also be insufficient. The ubiquitin plasmid-DNA should be increased to 3-4 μg per 10 cm cell culture dish.

The proteasome inhibitor MG132 may not be freshly prepared. MG132 stock solutions remain stable for 1-3 months at −20°C and once dissolved, they should be kept in small aliquot and protected from light.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Thomas Kietzmann (thomas.kietzmann@oulu.fi).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Carlos Vela-Rodriguez (carlos.velarodriguez@oulu.fi) for in vitro ubiquitination and Daniela Mennerich (daniela.mennerich@oulu.fi) for in vivo ubiquitination.

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed material transfer agreement.

Data and code availability

This work does not report original code.

Acknowledgments

We acknowledge funding from the Research Council of Finland (T.K., SA356920 and L.L., SA336449), Biocenter Oulu spearhead projects (L.L. and T.K.), and Jane and Aatos Erkko Foundation (L.L. and T.K.).

The use of the facilities and expertise of the Biocenter Oulu Structural Biology core facility (a member of Biocenter Finland, Instruct-ERIC Centre Finland and FINStruct), Proteomics and Protein Analysis core facility (a member of Biocenter Finland), and Biocenter Oulu sequencing center is gratefully acknowledged.

Author contributions

D.M. and C.V.-R. performed the experiments, analyzed the data, and wrote the original draft. L.L. and T.K. conceptualized and supervised the study, reviewed the manuscript, and obtained funding for the work.

Declaration of interests

The authors declare no competing interests.