Name: purification of ubiquitinated p53 proteins from mammalian cells
Uploaded: 2022-03-21T15:00:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Purification of Ubiquitinated p53 Proteins from Mammalian Cells at JoVE.com

This work was supported by a grant from the National Natural Science Foundation of China (81972624) to D.L.

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
<ol>
	<li>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 &#181;g/mL streptomycin). Maintain the cells at 37 &#176;C in a humidified incubator with 5% CO2. Split the cells every 2-3 days depending upon when they reach 80%-90% confluency.</li>
	<li>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. 
	&#8203;NOTE: HEK293T cells can also be used to purify ubiquitinated proteins due to their high transfection efficiency and protein expression level.</li>
</ol>
2. Plasmid transfection
<ol>
	<li>Add 1.5 mL of reduced serum medium in three 15 mL centrifuge tubes.</li>
	<li>Add plasmid DNA ready for transfection to each tube to make dilutions as follows. Tube 1: 1 &#181;g of Flag-p53 plasmid, 12 &#181;g of empty vector; Tube 2: 1 &#181;g of Flag-p53 plasmid, 3 &#181;g of His-HA-Ub (HH-Ub) plasmid, and 9 &#181;g of empty vector; Tube 3: 1 &#181;g of Flag-p53 plasmid, 3 &#181;g of HH-Ub plasmid, and 9 &#181;g of Mdm2 plasmid. Add a total of 0.2 &#181;g 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.</li>
	<li>Add 78 &#181;L 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).</li>
	<li>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 (&#181;g:&#181;L) for each transfection.</li>
	<li>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.</li>
	<li>Culture the cells in a humidified incubator at 37 &#176;C with 5% CO2. Replace the medium after 4-6 h and continue to culture the cells for 24-36 h.</li>
</ol>
3. Cell collection
<ol>
	<li>After 24-36 h, treat the cells with MG132 at a final concentration of 10 &#181;M 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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Purification of ubiquitinated proteins under nondenaturing conditions
<ol>
	<li>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.</li>
	<li>Add 800 &#181;L 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 &#176;C for 30 min.</li>
	<li>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 &#176;C for 30 min.</li>
	<li>Centrifuge the ultrasonicated samples at 8,000 x g for 20-30 min at 4 &#176;C. Transfer the supernatant to a new microcentrifuge tube.</li>
	<li>Aliquot 80 &#181;L (1/10) of the cell extract and mix with 20 &#181;L of 5x sodium dodecyl sulfate (SDS) loading buffer. Boil the samples at 98 &#176;C for 5 min, cool on ice for 2 min, and store at -20 &#176;C until use. Use these samples as the input group to monitor protein expression.</li>
	<li>Add 30 &#181;L of anti-Flag M2 antibody-conjugated (Flag/M2) beads to the remaining cell extracts and incubate on a rotator at 4 &#176;C for at least 4 h or overnight.</li>
	<li>Centrifuge at 1,500 x g for 2 min at 4 &#176;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.</li>
	<li>Centrifuge at 1,500 x g for 2 min at 4 &#176;C to collect the beads. Repeat step 4.7 for 4-6 times.</li>
	<li>Add 40 &#181;L of Flag peptides at a final concentration of 200 ng/&#181;L to the beads and incubate on a rotator at 4 &#176;C for 2 h to elute the bound proteins.</li>
	<li>Centrifuge at 1,500 x g for 5 min at 4 &#176;C. Transfer the supernatant to a new microcentrifuge tube.</li>
	<li>Add 10 &#181;L of 5x SDS loading buffer, boil the mixture at 98 &#176;C for 5 min, cool on ice for 2 min, and store at -20 &#176;C for Western blotting. Alternatively, store the eluate from step 4.10 directly at -80 &#176;C for other downstream experiments. 
	&#8203;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.</li>
</ol>
5. Purification of ubiquitinated proteins under denaturing conditions
<ol>
	<li>Add 1 mL of ice-cold PBS to the cell pellets obtained in step 3.3 and mix evenly. Aliquot 100 &#181;L (1/10 volume) of the cell suspension into a microcentrifuge tube as the input sample.</li>
	<li>Centrifuge at 700 x g for 5 min at 4 &#176;C to collect the cell pellets. Add 80 &#181;L 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.</li>
	<li>Centrifuge at 8,000 x g for 20-30 min at 4 &#176;C. Aliquot 80 &#181;L of the supernatant into a new microcentrifuge tube.</li>
	<li>Add 20 &#181;L of 5x SDS loading buffer to the supernatant, boil at 98 &#176;C for 5-10 min, cool on ice for 2 min, and store at -20 &#176;C until use.</li>
	<li>Centrifuge the remaining 900 &#181;L of the cell suspension at 700 x g for 5 min at 4 &#176;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 &#946;-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. &#946;-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.</li>
	<li>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.</li>
	<li>Add 30 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Elute bound proteins by incubating the beads with 40 &#181;L 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.</li>
	<li>Transfer the supernatant to a clean microcentrifuge tube, add 10 &#181;L of 5x SDS loading buffer, boil at 98 &#176;C for 5 min, cool on ice for 2 min, and store at -20 &#176;C for Western blotting. Alternatively, store the unprocessed eluate at -80 &#176;C for other downstream experiments.</li>
</ol>
6. Detection of purified ubiquitinated proteins by Western blotting
<ol>
	<li>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.</li>
	<li>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 &#176;C. Use the following antibodies for each set. All primary antibodies were used at a dilution of 1:1000.
	<ol>
		<li>Detect total p53 protein, including ubiquitinated p53, with an anti-p53 monoclonal antibody after immunoprecipitation with Flag/M2 agarose beads.</li>
		<li>Detect ubiquitinated p53 proteins with an anti-HA antibody or anti-ubiquitin antibody after immunoprecipitation with Flag/M2 agarose beads.</li>
		<li>Detect ubiquitinated p53 proteins with an anti-p53 monoclonal antibody after nickel-charged resin pulldown.</li>
		<li>Detect total ubiquitinated protein with an anti-HA antibody or anti-ubiquitin antibody after nickel-charged resin pulldown.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors declare no competing financial interests.

Ubiquitination plays a critical role in almost all physiological and pathological cellular processes2. In recent years, great progress has been made in understanding the molecular role of ubiquitin in signaling pathways and how changes in the ubiquitin system lead to different human diseases2. The purification of ubiquitinated proteins contributes to providing insight into the exact roles of ubiquitination in these processes. The mixtures of ubiquitin-conjugated proteins ca...

Ubiquitin is an evolutionarily conserved protein of 76 amino acids1,2,3. Ubiquitin covalently binds lysine residues on target proteins through cascades involving activating (E1), conjugating (E2), and ligase (E3) enzymes. Ubiquitin is first activated by the E1 enzyme and is then transferred to the E2 conjugating enzymes. Subsequently, E3 ubiquitin ligases interact with both ubiquitin-loaded E2 enzymes and substrate proteins and mediate the formation of an isopeptide bond between the C-terminal of ubiquitin and a lysine residue in the substrate1,2,3,4,5. Ubiquitination involves the attachment of ubiquitin moieties to lysine residues on substrate proteins or to itself, leading to protein monoubiquitination or polyubiquitination. This ubiquitination process occurs intracellularly in eukaryotes and regulates a large variety of biological processes. Ubiquitination results in the degradation of substrate proteins via the ubiquitin-proteasome system1,2,3,4,5. In addition, ubiquitination modulates protein subcellular localization, protein complex formation, and protein trafficking in cells3,5. Ubiquitin moieties ligated to substrate proteins can be removed by deubiquitinating enzymes (DUBs)6,7. Notably, the different ways in which ubiquitin chains are assembled provide a myriad of means to regulate various biological processes1,5. The exact roles of ubiquitination in regulating substrate protein function remain incompletely understood till now. The purification of ubiquitinated proteins contributes to the elucidation of the effects of protein ubiquitination on a variety of cellular processes.
The p53 protein is one of the most important tumor suppressor proteins and exhibits genetic mutations or inactivation in almost all human cancers8,9,10,11. p53 stability and activity are delicately regulated in vivo by posttranslational modifications, including ubiquitination, phosphorylation, acetylation, and methylation12,13. The p53 protein has a short half-life ranging from 6 min to 40 min in various cells, which results mainly from its polyubiquitination and subsequent proteasomal degradation10,12. Mouse double minute 2 (Mdm2) is an E3 ubiquitin ligase of p53 that binds to the N-terminus of p53 to inhibit its transcriptional activity12,14,15. Mdm2 promotes the polyubiquitination and proteasomal degradation of p53 to control its stability and induces monoubiquitination of p53 to facilitate its nuclear export12,14,15,16. Here, Mdm2-mediated p53 ubiquitination is used as an example to introduce a method for the purification of ubiquitinated proteins from mammalian cells in detail. The regulators that influence the ubiquitination status of target proteins can be identified using this in vivo ubiquitination assay when they are overexpressed or knocked down/knocked out in mammalian cells. In addition, ubiquitinated proteins can be used as substrates for in vitro deubiquitination assay. A high-throughput screening can be performed to identify specific DUBs for target proteins by incubating ubiquitinated substrates with individual DUBs. Ubiquitinated proteins may act as a scaffold to recruit downstream signaling proteins in cells. A ubiquitinated target protein complex can be purified by sequential immunoprecipitation under native purification conditions and identified by mass-spectrometry. The current protocol can be extensively used to investigate the cellular proteins regulated by ubiquitination.
Several methods have been established to purify ubiquitinated proteins, which include the use of affinity-tagged ubiquitin, ubiquitin antibodies, ubiquitin-binding proteins, and isolated ubiquitin-binding domains (UBDs)17. Here, we provide a protocol using affinity-tagged ubiquitin as a mediator to purify ubiquitinated proteins in mammalian cells. The use of poly-His-tagged ubiquitin offers advantages over the other methods. Ubiquitinated proteins are purified in the presence of strong denaturants, which reduces non-specific binding to nickel-charged resin by linearizing cellular proteins and disrupting protein-protein interactions. In contrast, the use of ubiquitin antibodies, ubiquitin-binding proteins, and isolated UBDs as mediators cannot effectively exclude binding partners from target protein because purification needs to be performed under less stringent conditions. Moreover, purification may also lead to increased binding of unrelated proteins using these mediators. In addition, there is a binding propensity for various ubiquitin linkage types as well as mono- and poly-ubiquitination by ubiquitin-binding proteins or isolated UBDs17. The use of poly-His-tagged ubiquitin contributes to pull down all cellular ubiquitinated proteins. Alternatively, the use of commercially available anti-Flag or anti-HA antibody-conjugated agarose make it easier to immunoprecipitate large-scale Flag- or HA-tagged target proteins under nondenaturing conditions. A second purification step, for example, by nickel-charged resin targeting poly-His-tagged ubiquitin, can be used to acquire ubiquitinated target proteins with a high purity for downstream experiments. Notably, an epitope tagging purification strategy can be adapted when a specific antibody cannot be acquired to immunoprecipitate target proteins effectively. Finally, purification of ubiquitinated proteins in mammalian cells, in comparison with purification in vitro, retains the ubiquitin linkage mode of target proteins under more physiological conditions.

<table><tbody><tr><td>&amp;beta;-mercaptoethanol</td><td>Sangon Biotech</td><td>M6250</td><td></td></tr><tr><td>Amersham ECL Mouse IgG, HRP-linked whole Ab (from sheep)</td><td>GE healthcare</td><td>NA931</td><td>Secondary antibdoy</td></tr><tr><td>Amersham ECL Rat IgG, HRP-linked whole Ab (from donkey)</td><td>GE healthcare</td><td>NA935</td><td>Secondary antibdoy</td></tr><tr><td>Anti-Flag M2 Affinity Gel</td><td>Sigma-Aldrich</td><td>A2220</td><td>FLAG/M2 beads</td></tr><tr><td>Anti-GFP monocolonal antibody</td><td>Santa cruz</td><td>sc-9996</td><td>Primary antibody</td></tr><tr><td>Anti-HA High Affinity</td><td>Roche</td><td>11867423001</td><td>Primary antibody</td></tr><tr><td>Anti-Mdm2 monocolonal antibody (SMP14)</td><td>Santa cruz</td><td>sc-965</td><td>Primary antibody</td></tr><tr><td>Anti-p53 monocolonal antibody (DO-1)</td><td>Santa cruz</td><td>sc-126</td><td>Primary antibody</td></tr><tr><td>EDTA</td><td>Sigma-Alddich</td><td>E5134</td><td>solvent</td></tr><tr><td>Fetal Bovine Serum</td><td>VivaCell</td><td>C04001-500</td><td>FBS</td></tr><tr><td>FLAG Peptide</td><td>Sigma-Alddich</td><td>F3290</td><td>Prepare elution buffer</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td>supplement</td></tr><tr><td>Guanidine-HCI</td><td>Sangon Biotech</td><td>A100287-0500</td><td>solvent</td></tr><tr><td>H1299</td><td>Stem Cell Bank, Chinese Academy of Sciences</td><td></td><td></td></tr><tr><td>Image Lab</td><td>Bio-rad</td><td></td><td>software</td></tr><tr><td>Immidazole</td><td>Sangon Biotech</td><td>A500529-0100</td><td>solvent</td></tr><tr><td>Immobilon Western Chemiluminescent HRP Substrate</td><td>Millipore</td><td>WBKLS0500</td><td></td></tr><tr><td>Lipofectamine 2000 reagents</td><td>Invitrogen</td><td>11668019</td><td>Transfection reagent</td></tr><tr><td>MG132</td><td>MedChemExpress</td><td>HY-13259</td><td>Proteasome inhibitor</td></tr><tr><td>Na2HPO4</td><td>Sangon Biotech</td><td>A501727-0500</td><td>solvent</td></tr><tr><td>NaCl</td><td>Sangon Biotech</td><td>A610476-0005</td><td>solvent</td></tr><tr><td>NaF</td><td>Sigma-Alddich</td><td>201154</td><td>solvent</td></tr><tr><td>NaH2PO4</td><td>Sangon Biotech</td><td>A501726-0500</td><td>solvent</td></tr><tr><td>Ni-NTA Agarose</td><td>QIAGEN</td><td>30230</td><td>nickel-charged resin</td></tr><tr><td>Nitrocellulose Blotting membrane</td><td>GE healthcare</td><td>10600002</td><td>0.45 &amp;micro;m pore size</td></tr><tr><td>Opti-MEM reduced serum medium</td><td>Gibco</td><td>31985-070</td><td>Transfection medium</td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-cv</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Sangon Biotech</td><td>E607011-0100</td><td>antibiotic</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8340</td><td></td></tr><tr><td>RPMI 1640</td><td>Biological Industries</td><td>01-100-1ACS</td><td>medium</td></tr><tr><td>Sarkosyl</td><td>Sigma-Alddich</td><td>L5777</td><td>solvent</td></tr><tr><td>SDS Loading Buffer</td><td>Beyotime</td><td>P0015L</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360-070</td><td>supplement</td></tr><tr><td>Tris-base</td><td>Sangon Biotech</td><td>A501492-0005</td><td>solvent</td></tr><tr><td>Tris-HCI</td><td>Sangon Biotech</td><td>A610103-0250</td><td>solvent</td></tr><tr><td>Triton X-100</td><td>Sangon Biotech</td><td>A110694-0500</td><td>reagent</td></tr><tr><td>Tween-20</td><td>Sangon Biotech</td><td>A100777-0500</td><td>supplement</td></tr><tr><td>Ultra High Sensitive Chemiluminescence Imaging System</td><td>Bio-rad</td><td></td><td>ChemiDoc XRS+</td></tr><tr><td>Urea</td><td>Sangon Biotech</td><td>A510907-0500</td><td>solvent</td></tr></tbody></table>

purification of ubiquitinated p53 proteins from mammalian cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The schematic diagram shows the Flag-tagged p53 (Flag-p53) and His/HA double-tagged ubiquitin (HH-Ub) proteins (Figure 1A). The procedures utilized to purify ubiquitinated proteins are summarized in Figure 1B. Poly-His-tagged ubiquitin can be ligated to target proteins in mammalian cells. Ubiquitinated proteins can be purified with Flag/M2 beads under nondenaturing conditions or by immobilized metal ion affinity chromatography (IMAC) under denaturing conditions ...

Watch this Scientific Journal Video about Purification of Ubiquitinated p53 Proteins from Mammalian Cells at JoVE.com

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

purification-of-ubiquitinated-p53-proteins-from-mammalian-cells

Research

JoVE Journal

Biochemistry

2.0K Views.  The Affiliated Zhangjiagang Hospital of Soochow University. The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Video: Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biology

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Immunology and Infection

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

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Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Detection of Protein Ubiquitination

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity