Purification of Ubiquitinated p53 Proteins from Mammalian Cells

NOTE: H1299 cells were kindly provided by the Stem Cell Bank, Chinese Academy of Sciences and were proven to be negative for mycoplasma contamination.

1. Cell culture

1. For the initial culture, place 1 x 10⁶ cells of human lung adenocarcinoma cell line, H1299 in a 10 cm Petri dish with 10-12 mL of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine additive, 1% sodium pyruvate, and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin). Maintain the cells at 37 °C in a humidified incubator with 5% CO₂. Split the cells every 2-3 days depending upon when they reach 80%-90% confluency.
2. One day before transfection, prepare 6-9 x 10⁶ cells for three Petri dishes and plate 2-3 x 10⁶ cells in a single 10 cm Petri dish containing 10-12 mL of medium to achieve a confluence of 70%-90% during transfection.
   ​NOTE: HEK293T cells can also be used to purify ubiquitinated proteins due to their high transfection efficiency and protein expression level.

2. Plasmid transfection

1. Add 1.5 mL of reduced serum medium in three 15 mL centrifuge tubes.
2. Add plasmid DNA ready for transfection to each tube to make dilutions as follows. Tube 1: 1 µg of Flag-p53 plasmid, 12 µg of empty vector; Tube 2: 1 µg of Flag-p53 plasmid, 3 µg of His-HA-Ub (HH-Ub) plasmid, and 9 µg of empty vector; Tube 3: 1 µg of Flag-p53 plasmid, 3 µg of HH-Ub plasmid, and 9 µg of Mdm2 plasmid. Add a total of 0.2 µg green fluorescent protein (GFP) plasmid to each tube to monitor the transfection efficiency.
   NOTE: A pilot experiment should be performed to determine the optimal amounts of plasmids needed to achieve efficient target protein expression in cells.
3. Add 78 µL of liposome transfection reagent to 4.5 mL of reduced serum medium in another centrifuge tube to dilute liposomes. Mix thoroughly by flicking the tube and allow to stand for 5 min at room temperature (RT).
4. Add 1.5 mL of the diluted liposome solution into each tube from step 2.2 containing 1.5 mL of the diluted DNA solution. Mix thoroughly and crosslink the plasmids with the liposomes for at least 20 min at RT. Use a plasmid DNA: liposome ratio of 1:2 (µg:µL) for each transfection.
5. Discard the original medium from the Petri dishes and add 9 mL of reduced serum medium into each dish. Add 3 mL of the liposome-DNA mixture to each dish and mix the solution by gently shaking the plate back and forth 3x, and then left and right 3x for even distribution of the mixture in the plate.
6. Culture the cells in a humidified incubator at 37 °C with 5% CO₂. Replace the medium after 4-6 h and continue to culture the cells for 24-36 h.

3. Cell collection

1. After 24-36 h, treat the cells with MG132 at a final concentration of 10 µM for 4-6 h.
   NOTE: MG132 is a peptide aldehyde that efficiently blocks the proteolytic activity of the 26S proteasome complex. The amounts of proteins ubiquitinated with lysine-48 (K48)-linked polyubiquitin chains can be increased after cells are treated with MG132 or other proteasome inhibitors.
2. Discard the medium, wash the cells 2x (taking care to not flush out the cells) with ice-cold phosphate buffered saline (PBS), and aspirate the cells with serological pipets.
3. Add 1 mL of PBS to the dish, remove the cells by scraping with a clean scraper, and transfer the cell suspension into microcentrifuge tubes. Centrifuge at 700 x g for 5 min to collect cell pellets.

4. Purification of ubiquitinated proteins under nondenaturing conditions

1. Prepare Flag lysis buffer (50 mM Tris-HCl (pH 7.9), 137 mM NaCl, 10 mM NaF, 1 mM ethylene diamine tetraacetic acid (EDTA), 1% Triton X-100, 0.2% sarkosyl, and 10% glycerol) freshly supplemented with protease inhibitor cocktail before use.
2. Add 800 µL of ice-cold Flag lysis buffer to the cells in each tube, mix the cells using a vortex oscillator or pipette gun, and then incubate the mixture on a rotator at 4 °C for 30 min.
3. Subject the mixture to 5-10 brief pulses of ultrasonication. Perform the ultrasonication on the ice at a sonication frequency of 20 kHZ and 80% amplitude with each pulse lasting for 1 s. Incubate the mixture on a rotator at 40 revolutions per minute (rpm) at 4 °C for 30 min.
4. Centrifuge the ultrasonicated samples at 8,000 x g for 20-30 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube.
5. Aliquot 80 µL (1/10) of the cell extract and mix with 20 µL of 5x sodium dodecyl sulfate (SDS) loading buffer. Boil the samples at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C until use. Use these samples as the input group to monitor protein expression.
6. Add 30 µL of anti-Flag M2 antibody-conjugated (Flag/M2) beads to the remaining cell extracts and incubate on a rotator at 4 °C for at least 4 h or overnight.
7. Centrifuge at 1,500 x g for 2 min at 4 °C to collect the beads. Add 1 mL of ice-cold Flag lysis buffer to the beads and mix by inverting the tube several times.
8. Centrifuge at 1,500 x g for 2 min at 4 °C to collect the beads. Repeat step 4.7 for 4-6 times.
9. Add 40 µL of Flag peptides at a final concentration of 200 ng/µL to the beads and incubate on a rotator at 4 °C for 2 h to elute the bound proteins.
10. Centrifuge at 1,500 x g for 5 min at 4 °C. Transfer the supernatant to a new microcentrifuge tube.
11. Add 10 µL of 5x SDS loading buffer, boil the mixture at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C for Western blotting. Alternatively, store the eluate from step 4.10 directly at -80 °C for other downstream experiments.
    ​NOTE: The amount of purified ubiquitinated proteins can be scaled up by increasing the number of cells and amounts of plasmids transfected. An epitope tagging strategy can be adapted to purify cellular complexes that bind specifically to ubiquitinated p53 under native purification conditions. By co-expressing Flag-p53, mdm2, and HA-ubiquitin, the ubiquitinated p53 protein complex may be immunoprecipitated by sequential Flag and HA antibody-conjugated agarose from mammalian cells. To keep binding partners of ubiquitinated p53 proteins, the less stringent BC100 lysis buffer (20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10% glycerol, 0.2 mM EDTA, 0.2% Triton X-100, and 1x protease inhibitor) should be used during the process of purification. The eluate from HA peptide is subjected to mass spectrometry to identify all partners that bind specifically to ubiquitinated p53.

5. Purification of ubiquitinated proteins under denaturing conditions

1. Add 1 mL of ice-cold PBS to the cell pellets obtained in step 3.3 and mix evenly. Aliquot 100 µL (1/10 volume) of the cell suspension into a microcentrifuge tube as the input sample.
2. Centrifuge at 700 x g for 5 min at 4 °C to collect the cell pellets. Add 80 µL of Flag lysis buffer to the input sample, mix the cells using a vortex oscillator or pipette gun, and lyse the cells on ice for 1 h.
3. Centrifuge at 8,000 x g for 20-30 min at 4 °C. Aliquot 80 µL of the supernatant into a new microcentrifuge tube.
4. Add 20 µL of 5x SDS loading buffer to the supernatant, boil at 98 °C for 5-10 min, cool on ice for 2 min, and store at -20 °C until use.
5. Centrifuge the remaining 900 µL of the cell suspension at 700 x g for 5 min at 4 °C to collect the cell pellet. Add 1 mL of ubiquitin buffer 1 (UB buffer 1; 6 M guanidine-HCI, 0.1 M Na₂HPO₄, 6.8 mM NaH₂PO₄, 10 mM Tris-HCI (pH 8.0), and 0.2% Triton X-100, freshly supplemented with 10 mM β-mercaptoethanol and 5 mM imidazole) to the cell pellets and mix several times by pipetting up and down to evenly distribute the cells.
   NOTE: The concentration of imidazole may vary from 5 mM to 20 mM to decrease nonspecific protein binding on the nickel-charged resin. Ubiquitin buffer contains guanidine hydrochloride, which inactivates both ligases and DUBs. β-mercaptoethanol is a clear, colorless liquid with a strong, unpleasant odor similar to rotten eggs. A high concentration solution will cause serious damage to the mucous membrane, upper respiratory tract, skin, and eyes. Wear gloves and goggles and operate in the fume hood.
6. Subject the cell lysates to 10-20 rounds of ultrasonication until the solution is no longer viscous. Perform the ultrasonication on the ice at a sonication frequency of 20 kHZ and 80% amplitude with each pulse lasting for 1 s. Centrifuge at 8,000 x g for 20-30 min at RT and transfer the supernatant to a new microcentrifuge tube.
7. Add 30 µL of nickel-charged resin to the supernatant and incubate on a rotator set at 15 rpm for 4 h or overnight at RT. Centrifuge at 1,500 x g for 2 min at RT to collect the beads.
8. Add 1 mL of UB buffer 1, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min at RT to collect the beads.
9. Add 1 mL of ubiquitin buffer 2 (UB buffer 2; 8 M urea, 0.1 M Na₂HPO₄, 6.8 mM NaH₂PO₄, 10 mM Tris-HCI (pH 8.0), and 0.2% Triton X-100) to the beads, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min to collect the beads. Repeat this once more.
10. To the beads, add 1 mL of PBS, incubate with rotation in a shaker for 10 min at RT, and centrifuge at 1,500 x g for 2 min at RT to collect the beads. Repeat this once more.
11. Elute bound proteins by incubating the beads with 40 µL of imidazole at a final concentration of 0.5 M for 1 h at RT. Centrifuge at 1,500 x g for 2 min at RT.
12. Transfer the supernatant to a clean microcentrifuge tube, add 10 µL of 5x SDS loading buffer, boil at 98 °C for 5 min, cool on ice for 2 min, and store at -20 °C for Western blotting. Alternatively, store the unprocessed eluate at -80 °C for other downstream experiments.

6. Detection of purified ubiquitinated proteins by Western blotting

1. Resolve the samples from steps 4.11 and 5.12 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transfer them to a nitrocellulose membrane by Western blotting to detect target proteins using the corresponding antibodies as described¹⁸.
2. In brief, incubate the membrane by immersing in 20 mL of blocking solution containing 5% defat milk powder in TBST buffer (15 mM Tris-HCI; pH = 7.6, 4.6 mM Tris-base, 150 mM NaCI, freshly supplemented with 0.1% Tween-20 before use) at RT for 1 h. Then, incubate the membrane with primary antibodies at RT for 2 h or overnight at 4 °C. Use the following antibodies for each set. All primary antibodies were used at a dilution of 1:1000.
   1. Detect total p53 protein, including ubiquitinated p53, with an anti-p53 monoclonal antibody after immunoprecipitation with Flag/M2 agarose beads.
   2. Detect ubiquitinated p53 proteins with an anti-HA antibody or anti-ubiquitin antibody after immunoprecipitation with Flag/M2 agarose beads.
   3. Detect ubiquitinated p53 proteins with an anti-p53 monoclonal antibody after nickel-charged resin pulldown.
   4. Detect total ubiquitinated protein with an anti-HA antibody or anti-ubiquitin antibody after nickel-charged resin pulldown.
3. Incubate the membrane with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h after washing 3x with TBST buffer for 5 min each. All secondary antibodies were used at a dilution of 1:3000. Then, wash the membrane with TBST buffer 3x for 5 min each with gentle agitation.
4. Start chemiluminescence imaging system to detect signals from Western blotting. Prepare the substrate solution for HRP by mixing solutions A and B at a ratio of 1:1.
5. Place the nitrocellulose membrane in the camera obscura face up and coat the substrate solution evenly on the membrane using a pipetting gun. Select the automatic exposure procedure to capture chemiluminescent signals and manually adjust the exposure time until an ideal signal is acquired.