Immune-Complex-Based In Vitro Deubiquitination Assay

Materials and reagents

Biological materials

1. HEK 293T/17 (American Type Culture Collection, CRL-11268)

Antibodies and plasmids

1. Pierce Anti-c-Myc agarose: agarose-conjugated anti-c-Myc (9E10) mouse monoclonal antibody (Pierce, catalog number 20168)

2. Purified anti-HA.11 Epitope Tag Antibody (clone 16B12) (BioLegend/Revvity, catalog number: 901502) (previously Covance, catalog number: MMS-101P)

3. Rabbit polyclonal Anti-c-Myc (A-14) antibody, 100 μg/mL (Santa Cruz, catalog number: Sc-789)

4. Rabbit polyclonal Anti-Flag antibody (Sigma, catalog number: F7425)

5. Goat anti-rabbit IgG (H + L), HRP-conjugated (KPL, catalog number: 074-1506)

6. Goat anti-mouse IgG (H + L), HRP-conjugated (KPL, catalog number: 074-1806)

7. pCDNA3.1-Myc-β-catenin, pCDNA3.1-Flag-USP17^WT, and pCDNA3.1-Flag-USP17^C89S, recently described [8]

8. pCDNA3.1-HA-ubiquitin plasmid, described in [10]

Reagents

1. DMEM Hi-glucose, L-glutamine, Na pyruvate (Wisent, catalog number: 319-005)

2. Fetal bovine serum (FBS), Premium, low endotoxin (Wisent, catalog number: 090-150, lot number: 112733)

3. Trypsine-EDTA (0.05%) (Wisent, catalog number: 325-542-CL)

4. Trypan Blue 0.4% solution (Wisent, catalog number: 609-130-EL)

5. Extreme MEM 1× with Earle’s salts, L-glutamine, and hormones (Wisent, catalog number: 390-005-CL)

6. Methanol 20 L (VWR, catalog number: CAMX0485-5)

7. Hydrochloric acid (HCl) concentrate, 10N ACS (certified) (Fisher, catalog number: SA49_6×100mL)

8. Sodium hydroxide (NaOH) solution (10N certified) (Fisher, catalog number: SS2551)

9. Acetic acid glacial (Fisher, catalog number: 351271500)

10. Anhydrous ethyl alcohol (Greenfield Global, catalog number: P016EAAN)

11. Contec^TM PREempt^TM concentrate disinfectant (Fisher Scientific, catalog number: 296366215)

12. Polyethylenimine, linear, MW 25000, transfection grade (PEI 25K^TM) (Polysciences, catalog number: 23966)

13. Glycerol (Wisent, catalog number: 800-040-LL)

14. Glycine (Wisent, catalog number: 800-045-IK)

15. Tris (hydroxymethyl) aminomethane (Wisent, catalog number: 600-125-IK)

16. Sodium chloride anhydrous (NaCl) (Wisent, catalog number: 600-082 IK)

17. Potassium chloride (KCl) (molecular biology, ≥99.0%) (Sigma, catalog number: P9541)

18. Sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O) (Sigma, catalog number: S9390)

19. Potassium phosphate monobasic (KH₂PO₄) (molecular biology, anhydrous) (Sigma, catalog number: 60218)

20. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma, catalog number: M2670)

21. Sodium fluoride (NaF) (Sigma, catalog number: S7920)

22. β-Glycerophosphate disodium salt hydrate (Sigma, catalog number: G6251)

23. Aprotinin from bovine lung (Roche, catalog number: 10236624001)

24. Pepstatin A from Streptomyces species (Roche, catalog number: 11359053001)

25. Sodium orthovanadate (Na₃VO₄) (Sigma, catalog number: 567540)

26. DL-Dithiothreitol (DTT) (Sigma, catalog number: D9779)

27. SDS 10% solution (Wisent, catalog number: 880-550-CL)

28. EDTA 0.5 M (pH 8.0) (Wisent, catalog number: 809-163 EL)

29. BSA standard 2 mg/mL (Thermo Scientific^TM Pierce^TM, catalog number: 23209)

30. Triton X-100 (Sigma, catalog number: X100-1L)

31. Tween-20 (Sigma, catalog number: P9416)

32. SDS (sodium dodecyl sulfate) (Wisent, catalog number: 800-100 CG)

33. Ponceau S (Sigma, catalog number: P3504-10G)

34. Sodium azide (NaN₃) (EM Science, MilliporeSigma, catalog number: SX0299-1)

35. Dry milk (Carnation, Skim Milk Fat-Free, 500 g)

36. Ammonium persulfate (APS) (Sigma, catalog number: A3678-100G)

37. Bromophenol blue (Bio-Rad, catalog number: 1610404)

38. TEMED (Sigma, catalog number: T9281-25ML)

39. DMSO (Sigma, catalog number: D8418)

40. Bortezomib (Sigma, catalog number: SKU 503140001)

41. Protein assay dye reagent concentrate (Bio-Rad, catalog number: 5000006)

42. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad, catalog number: 1610158)

43. Western Lightning ECL Pro 340 mL (Revvity; formerly, PerkinElmer’s Life Sciences and Diagnostics; catalog number: NEL121001EA)

44. Super Signal West Femto Maximum Sensitivity Substrate (Pierce, catalog number: 34095)

45. Protran^TM Premium Western blotting membrane, nitrocellulose, 0.2 μm (Amersham^TM, catalog number: GE10600001)

46. Parafilm^® M sealing film (VWR, catalog number: 52858-000)

47. Plastic film (Saran Wrap)

48. BLUelf Prestained Protein Ladder (Froggabio, catalog number: PM008-0500)

Note: For reagent storage conditions and expected shelf life, please refer to Supplementary File 1.

Solutions

1. 1× PBS buffer (see Recipes)

2. PEI (1 mg/mL) (see Recipes)

3. Bortezomib 10 mM (see Recipes)

4. NaCl 5 M (see Recipes)

5. MgCl₂ 2 M (see Recipes)

6. NaF 0.5 M (see Recipes)

7. β-Glycerophosphate 1 M (see Recipes)

8. Aprotinin 2 mg/mL (see Recipes)

9. Pepstatin A 1 mg/mL (see Recipes)

10. Na₃VO₄ 0.1 M (see Recipes)

11. DTT 1 M (see Recipes)

12. 10% Triton X-100 (see Recipes)

13. 1 M Tris-HCl pH 7.5 (see Recipes)

14. 1 M Tris-HCl pH 7.4 (see Recipes)

15. Hypotonic buffer + protease inhibitor cocktail (PIC) (see Recipes)

16. Lysis buffer (see Recipes)

17. DUBS assay buffer 5× (see Recipes)

18. 2% SDS in DUBS assay buffer (see Recipes)

19. DUBS assay buffer 1× (see Recipes)

20. 1.5 M Tris pH 8.8 (see Recipes)

21. 2 M Tris pH 6.8 (see Recipes)

22. 0.5 M Tris pH 6.8 (see Recipes)

23. 10% APS (see Recipes)

24. 30% Acrylamide 37.5:1 (see Recipes)

25. 10% SDS-PAGE (see Recipes)

26. 4% SDS-PAGE stacking gel (see Recipes)

27. Sample buffer 5× (see Recipes)

28. 10× Tris-glycine running buffer (see Recipes)

29. 10× transfer buffer (see Recipes)

30. Ponceau S solution (see Recipes)

31. 10% NaN₃ (see Recipes)

32. 10× TBS (see Recipes)

33. Stripping solution (see Recipes)

Recipes

1. 10× PBS buffer

Complete to 1 L with Milli-Q water to make 10×.

2. PEI (1 mg/mL)

Start with 50 mg of PEI + 45 mL of H₂O + 12 μL of HCl 10N. Dissolve completely, adding HCl if necessary, but do not drop the pH below 7.0. The final pH should be between 6.5 and 7.5. Adjust the volume to 50 mL. Once the PEI is dissolved and the volume is adjusted, filter-sterilize the solution (0.22 μm), aliquot, and freeze at -20 °C. Stable at 4 °C for 1 month; it may be freeze-thawed five times.

3. Bortezomib 10 mM

To prepare a 10 mM stock solution from a 2.5 mg vial of bortezomib (molecular weight: 384.24 g/mol), add 651 μL of DMSO. Aliquot the stock solution into single-use vials and store at -20 °C for up to 6 months.

4. NaCl 5 M

Dissolve completely in 75 mL of Milli-Q water, then dilute to a final volume of 100 mL.

5. MgCl₂ 2 M

Dissolve completely in 80 mL of Milli-Q water, then dilute to a final volume of 100 mL. Autoclave or filter through 0.22 μm.

6. NaF 0.5 M

Dissolve completely in 40 mL of Milli-Q water, then dilute to a final volume of 50 mL. Keep at 4 °C.

7. β-Glycerophosphate 1 M

Dissolve completely in 80 mL of Milli-Q water, then dilute to a final volume of 100 mL.

8. Aprotinin 2 mg/mL

Dissolve the contents of the vial in 4 mL of Milli-Q water, then bring the final volume to 5 mL. Divide into aliquots of 75 μL and use at a 1/1,000 dilution (2 μg/mL). Store stocks at -80 °C.

9. Pepstatin A 1 mg/mL

Dissolve the contents of the vial in 10 mL of 100% ethanol and rotate overnight at 4 °C. Then, prepare 75 μL aliquots and use at a 1/1,000 dilution (1 μg/mL). Store aliquots at -80 °C.

10. Na₃VO₄ 0.1 M

a. Dissolve 0.92 g of sodium orthovanadate in 20 mL of Milli-Q water in a 50 mL tube.

b. Adjust the pH to 10 (the solution will turn yellow).

c. Boil the solution in a water bath for 5–10 min (until the solution becomes colorless).

d. Let the solution sit until it cools down to room temperature (RT).

e. Readjust pH to 10.0.

f. Repeat steps c–e until the pH is stable at 10.0 and the solution is colorless.

g. Fill up to 50 mL with Milli-Q H₂O to obtain 0.1 M.

h. Prepare 750 μL aliquots and freeze stocks at -80 °C. Refreeze tubes at -20 °C for routine assay.

11. DTT 1 M

Dissolve 248.5 mg of DTT powder into 1.5 mL of Milli-Q water. Once completely dissolved, add water to bring the final volume to 2 mL. Prepare aliquots of 100 μL. Keep at -80 °C. Once thawed, refreeze at -20 °C 3–4 times.

12. 10% Triton X-100

With a P1000 tip, take 1 mL of Triton X-100 by releasing the push button of the pipettor very slowly. Wipe the excess viscous solution from the tip with a Kimtech lint-free wipe, add to the water, and rinse the contents of the tip with a few up/down strokes as needed. Store at 4 °C.

13. 1 M Tris pH 7.4

Dissolve Tris in approximately 800 mL of MilliQ water. Adjust pH to 7.4 using 10N HCl. Store at 4 °C to cool the solution. Once the solution reaches 4 °C, readjust the pH to 7.4, then fill to 1 L and store at 4 °C.

14. 1 M Tris pH 7.5

Dissolve Tris in approximately 800 mL of Milli-Q water. Adjust pH to 7.5 using 10N HCl. Store at 4 °C to cool the solution. Once the solution reaches 4 °C, readjust the pH to 7.5, then fill to 1 L and store at 4 °C.

15. Hypotonic buffer + PIC

Prepare the buffer freshly as indicated and keep it on ice. The buffer may be frozen.

16. Lysis buffer

Add all the reagents to the Milli-Q water, mix, and keep on ice; no protease inhibitors are added.

17. DUBS assay buffer 5×

Add all the reagents to the Milli-Q water, mix, and keep on ice. Freeze the stock at -20 °C.

18. 2% SDS in DUBS assay buffer

Dilute the 5× DUBS assay buffer with water to 1×. Add the SDS, prepare freshly, and use at RT to avoid SDS precipitation.

19. DUBS assay buffer 1×

Add all the reagents to the Milli-Q water, mix, and keep on ice. No protease inhibitor is added.

20. 1.5 M Tris pH 8.8

Dissolve 181.7 g of Tris base in 850 mL of Milli-Q water. Adjust the pH to 8.8 using concentrated HCl (approximately 28 mL). Readjust the pH to 8.8 at room temperature. Add H₂O to 1 L. Store the buffer at 4 °C.

21. 2 M Tris pH 6.8

Dissolve 121.1 g of Tris in 400 mL of Milli-Q water. Adjust the pH to 6.8 using concentrated HCl (approximately 40–45 mL). Readjust the pH to 6.8 at room temperature. Add Milli-Q H₂O to 500 mL. Store the buffer at 4 °C.

22. 0.5 M Tris pH 6.8

Dissolve 60.5 g of Tris base in 850 mL of Milli-Q water. Adjust the pH to 6.8 using concentrated HCl (approximately 40– 45 mL). Readjust the pH to 6.8 at room temperature. Add Milli-Q H₂O to 1,000 mL. Store the buffer at 4 °C.

23. 10% APS (w/v)

Dissolve 1 g of APS in 10 mL of Milli-Q H₂O. Store at 4 °C. Stable for three months.

24. 30% Acrylamide 37.5:1

Add 362 mL of Milli-Q water directly into the bottle, then add a magnetic stirrer (small to medium size) and mix on the magnetic plate for 30 min on medium speed. Once dissolved completely, filter through a 0.45 μm filter unit.

Note: To minimize contact with acrylamide powder or solution, please follow the company protocol. The recommended shelf life of the solution is 1 month at 4 °C in a dark bottle.

Acrylamide, whether in powder or solution, is toxic. The proper use of gloves and masks is highly recommended.

25. 10% SDS-PAGE

In a clean tube, add all the solutions except TEMED and APS. At the last minute, when the glass setup is ready, add these last two reagents. Mix with a pipette controller without any bubbles; up/down movements may be performed. Then, disperse onto the casting setup, moving from one side to the other (left to right). Gently add 500 μL of Milli-Q water on top of the acrylamide; there is no need to use any saturated solvent to delimit the top of the gel. Let it polymerize for at least 1 h at RT.

26. 4% SDS-PAGE stacking gel

Proceed as for Recipe 25; let it polymerize for at least 30 min.

27. Sample buffer 5×

*Manipulate the solution under the chemical hood when adding -mercaptoethanol and after.

In a 50 mL conical tube, add Tris, glycerol, SDS, β-mercaptoethanol, and Milli-Q water, then slowly agitate (rocker) until the solution is clear at RT (overnight).

Weigh the BPB and add it to the 50 mL tube; close tightly. Incubate in a closed container filled with warm tap water. Leave the tube on the rocker until the solution is homogeneous. Aliquot in 500 μL and keep stock at -20 °C.

28. 10× Tris-glycine running buffer

Wear a mask and gloves, as SDS is volatile and harmful. In a large beaker, add 600 mL of Milli-Q water, Tris base, glycine, and SDS slowly. Protect with plastic film and agitate on a magnetic stirrer overnight or until dissolved at RT. Complete to 1 L by adding water without creating bubbles.

Note: Mixing the solution produces a lot of foam. Pour gently into the stock bottle.

Prepare 400 mL of 1× SDS running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS) per montage when ready for electrophoresis. Dilute 40 mL of 10× with 360 mL of Milli-Q water; mix thoroughly.

29. 10× Transfer buffer 1 L

In a 1 L beaker, add Tris and glycine to 350 mL of Milli-Q water. Protect with a plastic film; agitate at RT on a magnetic stirrer until dissolved. Complete to 1 L by adding water. Store at 4 °C.

When ready to transfer, dilute the transfer buffer by adding 100 mL of 10× transfer buffer to 700 mL of cold water, then add 200 mL of methanol (under the chemical hood), mix, and keep at 4 °C. The buffer may be reused once.

30. Ponceau S solution

In a 475 mL bottle of Milli-Q water, add 0.5 g of Ponceau S and agitate on a magnetic stirrer until fully dissolved. Finally, add 25 mL of acetic acid slowly, close the bottle, keep at RT, and reuse this solution until the staining becomes faint.

31. 10% NaN3 (w/v)

Dissolve 1 g of sodium azide in 10 mL of 1× PBS. Store at 4 °C. Use at 0.02% final concentration (20 μL of NaN₃ in 10 mL of first-antibody dilution).

32. 10× TBS 2 L

Before filling to 2 L with Milli-Q water, adjust pH to 8.0. This requires approximately 22 mL of 10N HCl. When pH has been adjusted to 8.0, wait 30 min and take another measurement. If the pH is still at 8.0, fill the solution to 2 L with Milli-Q water. If not, adjust it to 8.0 and wait another 30 min to make sure the pH is stable.

Prepare 1× TBS + Tween-20 (0.1%) (TBST) for all western blot experiments. Dilute 200 mL of 10× TBS in 1,798 mL of Milli-Q water and mix on a magnetic stirrer. With a 5 mL pipette, very slowly retrieve 2 mL of Tween-20 from the 100% solution; wait until the pipettor stops aspirating to the 2 mL volume. With a clean wipe tissue (Kimtech), remove any excess Tween-20 from the outside of the 5 mL pipette, then dispense the 2 mL volume into the solution with many up-and-down strokes until none remains. Continue mixing for at least 15 min.

33. Stripping buffer

Prepare freshly. Add the mercaptoethanol at the last minute, under the chemical hood. Dispose of the waste accordingly.

Laboratory supplies

1. Cell scraper (M) (Sarstedt, catalog number: 83.3951)

2. Four-channel alarm timer with clock (VWR, catalog number: 62344-641)

3. Petri dishes, 10 and 15 cm (Sarstedt, catalog numbers: 83.3902, 83.3903)

4. Pipettes, 5, 10, and 25 mL (Sarstedt, catalog numbers: 86.1253.001, 86.1254.001, 86.1685.001)

5. Tips, 10, 200, 1,000 μL (Ultident, catalog numbers: 87-A10, 87-A200C, 87-A1000B)

6. Glass Pasteur pipette 9 po (VWR, catalog number: 14673-043)

7. Blot paper (Bio-Rad, catalog number: 1650921)

8. Nitrocellulose membrane, 0.45 μm (Bio-Rad, catalog number: 1620115)

9. Kimtech lint-free wipe (Ultident, catalog number: 14-05511)

10. Microplate, clear 96-well, flat-bottom, with cover (Corning, catalog number: 3370)

11. 13 mL × 100 mm borosilicate tubes (Fisherbrand, catalog number: 14-961-27)

12. Microcentrifuge tubes 1.7 mL (Froggabio, catalog number: LMCT1.7B10)

13. Microcentrifuge tubes 5 mL snap cap (Froggabio, catalog number: LMCT5.OB10)

14. Conical centrifuge tubes, 15 and 50 mL (Froggabio, catalog numbers: TB15-500, TB50-500)

15. Filtration unit (500 mL), 0.45 and 0.22 μm (Sarstedt, catalog numbers: 83.3941, 83.3941.001)

16. Polystyrene rectangular dish with four compartments (Thermo Fisher Scientific, catalog number: 267061)

17. Antibody incubation boxes (Servicebio, catalog numbers: G6026-4, G6025-2, and G6020-9)

18. Filter forceps (Sigma, catalog number: XX6200006P)

19. Multiflex gel loading tip (ESBE, catalog number: SRS-2848)

Equipment

1. Hemacytometer (Sigma, model: Bright-Line)

2. Benchtop centrifuge (Sorvall, model: Legend RT)

3. Pipette controller (BrandTech, model: Accu-jet Pro)

4. Pipetman (Gilson, model: P2, P20, P200, P1000)

5. Microplate reader (BioTek, model: SynergyH1)

6. Mini centrifuge with 2 rotors (Benchmark Scientific, catalog number: Z742582)

7. Vortex (MBI-Scientific Industries SI, model: Genie 2)

8. Antibody incubation box (Servicebio Inc, catalog numbers: G6026-4, G6025-2, G6020-9)

9. Rectangular 4-well plate with cover (ThermoFisher, catalog number: 267061)

10. pH meter (Accumet, model: AB150)

11. Isotemp Magnetic Hotplate (Fisher Scientific, model: 11-600-100SH)

12. Dry Bath Incubator BenchMark (Sigma, catalog number: Z742509)

13. Refrigerated centrifuge (Eppendorf, model: 5417R)

14. Tube rotator (Fisher Scientific, model: 346 Chemistry Hematology Mixer test Tube rotator)

15. Hood Class II Type A2 (ESCO, model: LA2-6A2)

16. Cell incubator (Heraeus HERAcell 150i, catalog number: SVH-51022394)

17. Inverted microscope (Hund, model: Wilovert S)

18. Vacusafe (aspiration system) (Mandel, catalog number: TM-158310)

19. Molecular Image ChemiDoc with Universal Hood II (Bio-Rad, model: XRS+)

20. Refrigerated Incubator Max-Q 6000-7 (Barnstead, catalog number: BDI-SHKE6000-7)

21. Freezer -80 °C (REVCO, model: ULTIMA II ULT2586-9S)

22. Freezer -20 °C, 18 cu. ft. refrigerator (Frigidaire, model: FFTR1817LW)

23. Mini-PROTEAN Tetra Electrophoresis system (Bio-Rad, catalog number: 1658006)

24. Criterion^TM Blotter with plate electrode (Bio-Rad, catalog number: 1704070)

25. Power supply (Bio-Rad, model: Power PAC 200)

26. Eppendorf Thermomixer Compact (Sigma, catalog number: T1317)

27. Precision reciprocating digital water bath (Thermo Scientific, catalog number: 2870)

28. Rocker dual platform (Bio-Rad, catalog number: 1660709)

29. Digital dry bath (Sigma/Milipore, catalog number: Z742505)

30. Balances (Mettler Toledo, model: PG2002-S and AG204 Delta Range)

31. Spectrophotometer UV visible (GE Amersham, model: Ultrospec 2100 pro)

Software and datasets

1. Image Lab, version 6.1 (Bio-Rad)

Procedure