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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#946;-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>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 &#181;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

1.9K 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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Identification of Plant Ice-binding Proteins Through Assessment of Ice-recrystallization Inhibition and Isolation Using Ice-affinity Purification

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In plants, freeze damage occurs when extracellular ice crystals grow, resulting in the rupture of plasma membranes and possible cell death. Adsorption of IBPs to ice crystals restricts further growth by a process known as ice-recrystallization inhibition (IRI), thereby reducing cellular damage. IBPs also demonstrate the ability to depress the freezing point of a solution below the equilibrium melting point, a property known as thermal hysteresis (TH) activity. These protective properties have raised interest in the identification of novel IBPs due to their potential use in industrial, medical and agricultural applications. This paper describes the identification of plant IBPs through 1) the induction and extraction of IBPs in plant tissue, 2) the screening of extracts for IRI activity, and 3) the isolation and purification of IBPs. Following the induction of IBPs by low temperature exposure, extracts are tested for IRI activity using a &#39;splat assay&#39;, which allows the observation of ice crystal growth using a standard light microscope. This assay requires a low protein concentration and generates results that are quickly obtained and easily interpreted, providing an initial screen for ice binding activity. IBPs can then be isolated from contaminating proteins by utilizing the property of IBPs to adsorb to ice, through a technique called &#39;ice-affinity purification&#39;. Using cell lysates collected from plant extracts, an ice hemisphere can be slowly grown on a brass probe. This incorporates IBPs into the crystalline structure of the polycrystalline ice. Requiring no a priori biochemical or structural knowledge of the IBP, this method allows for recovery of active protein. Ice-purified protein fractions can be used for downstream applications including the identification of peptide sequences by mass spectrometry and the biochemical analysis of native proteins.

This paper outlines the identification of ice-binding proteins from freeze-tolerant plants through the assessment of ice-recrystallization inhibition activity and subsequent isolation of native IBPs using ice-affinity purification.

Ice-binding proteins (IBPs) belong to a family of stress-induced proteins that are synthesized by certain organisms exposed to subzero temperatures. In ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...