Name: Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model (Video) | JoVE Journal
Uploaded: 2026-04-17T14:05:00.000+00:00
Duration: 9 min
Description: Learn two cell lysis methods for detecting ubiquitination in mammalian cells using SMAD2/SMURF2 as a model. This protocol compares ice-bath and heat treatment approaches to help researchers select optimal techniques.

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5 minutes

This research study was supported by the National Natural Science Foundation of China, Nos. 82370647 (W.H.), Nos. 82170642 (J-X.Z), Nos. 81801923 (X-F.Z), Nos. 81670575 (J-X.Z), Nos.81570570 (W.H.) and Nos. 81070355 (J-X.Z), and the Program of HUST Academic Frontier Youth Team, Huazhong University of Science and Technology (2018QYTD02). The Open Foundation of Hubei Key Laboratory of Regenerative Medicine and Multi-disciplinary Translational Research (2022zsyx003). The National Key Research and Development Program of China (2024YFA1107601).

1. Preparation of lysis buffer and reagents
NOTE: Prepare all buffers at the indicated concentrations. Follow the manufacturer&#39;s instructions for reagent powders to avoid concentration discrepancies.
<ol>
	<li>Prepare lysis buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA (pH 8.0), and 1% Triton X-100. Adjust the NaCl concentration between 50-150 mM if required. Prepare all buffers using double-distilled H&#8322;O.</li>
	<li>Prepare working solution by supplementing lysis buffer with 1% protease inhibitor cocktail (without EDTA). Mix thoroughly and keep on ice until use.</li>
	<li>Prepare 10% SDS by dissolving 1 g of SDS powder in 10 mL of PBS. Mix until fully dissolved. Store at room temperature.</li>
	<li>Prepare 5&#215; protein loading buffer containing 250 mM Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 0.5% bromophenol blue, and 5% &#946;-mercaptoethanol. Dissolve components in double-distilled H2O. Mix thoroughly before use. 
	NOTE: Handle SDS and &#946;-mercaptoethanol in a fume hood while wearing appropriate protective equipment. Dispose of contaminated materials according to institutional chemical waste guidelines.</li>
</ol>
2. Preparation of cells
<ol>
	<li>Maintain HEK293T cells in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Incubate at 37 &#176;C with 5% CO&#8322; under sterile conditions. Passage cells at approximately 90% confluency.</li>
	<li>&#160;Seed cells in 6-well plates at 5 &#215; 10&#8309; cells per well. Pool cells from two wells to generate one sample.</li>
	<li>Prepare transfection mixtures to represent 10% of the total culture volume and use SMAD2 ubiquitination as the experimental model. Combine 1 &#181;g of HA-Ub plasmid, 1 &#181;g of FLAG-SMAD2 plasmid, and 0.5 &#181;g of empty vector or MYC-SMURF2 plasmid in 100 &#181;L of serum-free DMEM. In a separate tube, dilute 3.75 &#181;L of transfection reagent in 100 &#181;L of serum-free DMEM. Add the reagent mixture dropwise to the plasmid solution, incubate for 15 min at room temperature, and distribute evenly onto cells.</li>
	<li>Replace the transfection medium after 6-8 h with 2 mL of fresh complete DMEM per well. Incubate cells for 48 h.</li>
	<li>Treat cells with 5 &#181;M MG132 for 12 h before harvesting. 
	NOTE: Handle MG132 in a biosafety cabinet while wearing gloves. Dispose of contaminated materials according to institutional safety procedures.</li>
</ol>
3. Cell collection
<ol>
	<li>Harvest cells at 90-100% confluency (~3 &#215; 10&#8310; cells). Aspirate medium and wash each well with 1 mL of prechilled PBS. Remove PBS completely.</li>
	<li>Add 1 mL of PBS to each well and gently resuspend cells. Transfer the suspension to a 1.5 mL tube and place on ice.</li>
	<li>Centrifuge at 380 &#215; g for 5 min at 4 &#176;C. Remove the supernatant and retain the pellet.</li>
</ol>
4. Agarose bead preparation
<ol>
	<li>Transfer 50 &#181;L of Protein A/G agarose beads per sample to a new tube. Wash beads 3x with 1 mL of lysis buffer. Centrifuge at 100 &#215; g for 1 min at 4 &#176;C between washes.</li>
	<li>Remove residual buffer without disturbing the pellet. Add 100 &#181;L of working solution and 5 &#181;L of anti-FLAG antibody. Incubate for 6 h at 4 &#176;C with gentle rotation.</li>
</ol>
5. Cell lysis
<ol>
	<li>Ice-bath method
	<ol>
		<li>Add 500 &#181;L of prechilled working solution to the pellet. Incubate on ice for 30 min.</li>
		<li>Sonicate at 2 W using 4 s on/4 s off cycles for five repetitions. Keep samples on ice during sonication. Return samples to ice for the remainder of the incubation. 
		&#8203;NOTE: For weak protein interactions, reduce NaCl concentration to 50-75 mM and omit sonication.</li>
	</ol>
	</li>
	<li>Heat-treatment method
	<ol>
		<li>Add 45 &#181;L of working solution to the pellet. Add 5 &#181;L of 10% SDS while mixing continuously.</li>
		<li>Vortex briefly and heat at 95 &#176;C until the suspension becomes transparent and fully liquefied. Invert intermittently to ensure uniform denaturation.</li>
		<li>Add 450 &#181;L of working solution. Sonicate at 2 W using 4 s on/4 s off cycles for five repetitions while keeping samples on ice. 
		NOTE: Ensure final SDS concentration remains &#60;0.1%. Confirm complete lysis by verifying disappearance of visible pellet and uniform solution clarity.</li>
	</ol>
	</li>
</ol>
6. Immunoprecipitation and ubiquitination detection
<ol>
	<li>Centrifuge lysates at 13,680 &#215; g for 15 min at 4 &#176;C. Transfer supernatant to a new tube.</li>
	<li>Reserve 60 &#181;L of lysate and add 15 &#181;L of 5&#215; loading buffer. Heat at 95 &#176;C for 15 min and label as input.</li>
	<li>Add 100 &#181;L of antibody-coupled beads to the remaining lysate (final volume 540 &#181;L). Incubate at 4 &#176;C for 8-12 h with rotation.</li>
	<li>Centrifuge beads at 100 &#215; g for 1 min at 4 &#176;C and discard supernatant. Wash beads 3x with lysis buffer. Add 75 &#181;L of 1&#215; SDS loading buffer and heat at 95 &#176;C for 15 min to elute proteins. Label as IP.</li>
	<li>Detect ubiquitinated proteins by western blot according to a standard JoVE protocol. 
	NOTE: Refer to the western blot protocol29 for detailed transfer and detection procedures. Full-length uncropped blots with molecular weight markers are provided in Supplemental File 1.</li>
</ol>

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</ol>

The authors have no conflicts of interest to declare.

This study presents and compares two detailed protein lysis protocols specifically optimized for detecting E3 ligase-mediated substrate ubiquitination. To ensure detectability, we used plasmid-based overexpression (e.g., tagged SMAD2, SMURF2, or Ub) in cells, which provided sufficient protein levels to visualize specific ubiquitination signals, critical for validating E3 ligase-substrate interactions in this study. Prior to cell harvesting, MG132, a potent proteasome inhibitor, was added to block the proteasome-mediated ...

Ubiquitination, an indispensable post-translational modification, is a critical regulator of intracellular protein functions1. The ubiquitin-proteasome system (UPS), which mediates the degradation of over 80% of proteins in eukaryotes, is composed of ubiquitin (Ub), E1 activating enzymes, E2 conjugating enzymes, E3 ligases, deubiquitylating enzymes (DUBs), and the 26S proteasome2. This system regulates protein turnover with high specificity, thereby controlling essential cellular processes such as cell cycle progression3, signal transduction4,5, and stress responses6,7. Ubiquitination is a multi-step enzymatic process. E1 hydrolyzes ATP to adenylate the C-terminus of Ub, forming a Ub-AMP intermediate. Then, the activated Ub is transferred from E1 to the active site cysteine of an E2 conjugating enzyme, forming a thioester bond. Finally, an E3 ligase recruits both the E2-Ub complex and a target protein, catalyzing the transfer of Ub from E2 to a lysine residue on the substrate8.
Ubiquitin can form diverse polyubiquitin chains through linkage at seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) or Met1(M1)4. Each linkage type dictates distinct biological outcomes: K48-linked chains primarily target proteins for proteasomal degradation9, K63-linked chains regulate signaling and DNA repair10,11, and M1-linked chains are critical for innate immune responses12. Ubiquitination and deubiquitination are pivotal post-translational modification processes that govern diverse cellular events, ranging from protein degradation to signal transduction. For instance, in colorectal cancer, aberrant activation of MDM2-mediated p53 ubiquitination leads to excessive degradation of p53, impairing its ability to induce cell cycle arrest and apoptosis, thereby driving tumor progression and chemoresistance13,14. In hepatic ischemia-reperfusion injury, OTUD1 stabilizes NRF2 by removing its K48-linked polyubiquitination, thereby ameliorating intracellular oxidative stress levels15,16. A range of proteasome inhibitors, such as bortezomib, carfilzomib, and ixazomib, have progressively entered clinical practice for diseases such as multiple myeloma17,18,19.
Currently, there are numerous methods for detecting ubiquitinated proteins, including western blotting, radioactivity-based assays, and fluorescence assays20. However, in scientific research, the western blot remains the preferred method for the detection of protein ubiquitination due to its relatively rapid and straightforward protocol. Traditional methods for detecting ubiquitinated proteins are similar to co-immunoprecipitation (co-IP) technology: they use a lysis buffer to incubate and lyse cell pellets at low temperature to release proteins, then use antibodies to detect the ubiquitination of substrate proteins21,22. However, this experimental protocol often requires a large number of cells. Additionally, because the relatively mild lysis method at low temperature cannot completely disrupt protein interactions, this increases the likelihood of false positives in detected ubiquitination. Heating and the use of SDS can significantly disrupt protein structure, inducing their complete denaturation, thereby effectively reducing false positive results caused by protein-protein interactions23,24,25. Similarly, cells undergo sufficient lysis upon heating and SDS treatment, releasing more proteins and, correspondingly, reducing the amount of cells required for the experiment. In summary, both the ice-bath method and the heat-treatment method lyse cells and detect the ubiquitination levels of substrate proteins via antibody incubation. However, the heating method results in greater cell lysis and stronger protein denaturation.
TGF-&#946; plays a dual role in the pathophysiological processes of various diseases26,27. SMAD2 is a critical downstream transcription factor of TGF-&#946;. SMURF2-mediated ubiquitination of SMAD2 targets it for proteasomal degradation, thereby fine-tuning TGF-&#946; signaling intensity28. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model. Here, protein overexpression in HEK293T cells was induced by transient transfection with HA-Ub, FLAG-SMAD2, and MYC-SMURF2 plasmids. The cells were lysed using the ice-bath method and the heat-treatment method, respectively. Ubiquitinated proteins were detected by Western blot analysis. We found that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, and the heat-treatment method might make it easier to detect ubiquitination of SMAD2. This protocol provides a new alternative for detecting ubiquitinated proteins. Furthermore, for certain substrate proteins strongly associated with ubiquitination, the heat-treatment method achieves better protein lysis efficiency, thereby enabling easier detection of ubiquitination.

<table><tbody><tr><td>1.5 mL microtube</td><td>Servicebio</td><td>EP-150-M</td><td></td></tr><tr><td>100 mm TC-treated cultured dish</td><td>Servicebio</td><td>CCD-100</td><td></td></tr><tr><td>6-well disk</td><td>Servicebio</td><td>CCP-6H</td><td></td></tr><tr><td>Anti-FLAG antibodies</td><td>Proteintech</td><td>20543-1-AP</td><td>dilution used 1:200 for IP 1:10000 for WB</td></tr><tr><td>Anti-GAPDH antibodies</td><td>Proteintech</td><td>MA5-15738</td><td>dilution used 1:10000</td></tr><tr><td>Anti-HA antibodies</td><td>Proteintech</td><td>51064-2-AP</td><td>dilution used 1:10000</td></tr><tr><td>Anti-IgG antibodies</td><td>CST</td><td>#3900</td><td>dilution used 1:200 for IP</td></tr><tr><td>Anti-MYC antibodies</td><td>Proteintech</td><td>60003-2-Ig</td><td>dilution used 1:10000</td></tr><tr><td>Anti-SMAD2 antibodies</td><td>CST</td><td>#5339</td><td>dilution used 1:200 for IP 1:2000 for WB</td></tr><tr><td>Bromophenol blue</td><td>Servicebio</td><td>GC307006-5g</td><td></td></tr><tr><td>EDTA (pH=8.0)</td><td>Servicebio</td><td>G1207-1L</td><td></td></tr><tr><td>FBS</td><td>ABW</td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sinopharm</td><td>10010618</td><td></td></tr><tr><td>Heating/Cooling Dry Bath</td><td>Guocheng</td><td></td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>High-glucose DMEM</td><td>Servicebio</td><td>G4515-500ML</td><td></td></tr><tr><td>HRP-conjugated Goat anti-Mouse IgG Light Chain&amp;nbsp;</td><td>Abclonal</td><td>AS062</td><td>dilution used 1:5000</td></tr><tr><td>HRP-conjugated Mouse anti-Rabbit IgG Light Chain&amp;nbsp;</td><td>Abclonal</td><td>AS061</td><td>dilution used 1:10000</td></tr><tr><td>Lipo8000</td><td>Beyotime</td><td>C0533</td><td></td></tr><tr><td>MG132</td><td>Aladdin</td><td>M421264</td><td></td></tr><tr><td>Microcentrifuge</td><td>Labgic</td><td>CF1524R</td><td></td></tr><tr><td>NaCl powder</td><td>Servicebio</td><td>GC102004</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Servicebio</td><td>G4202-500ML</td><td></td></tr><tr><td>pCMV-FLAG-SMAD2</td><td></td><td></td><td>For mammalian expression</td></tr><tr><td>pCMV-HA-Ub</td><td></td><td></td><td>For mammalian expression</td></tr><tr><td>pCMV-MYC</td><td></td><td></td><td>For mammalian expression</td></tr><tr><td>pCMV-MYC-SMURF2</td><td></td><td></td><td>For mammalian expression</td></tr><tr><td>Penicillin-streptomycin</td><td>Servicebio</td><td>G4003-100ML</td><td></td></tr><tr><td>Protease inhibitor cocktail without EDTA</td><td>TargetMol</td><td>C0001</td><td></td></tr><tr><td>Protein A/G agarose beads</td><td>Beyotime</td><td>P2017</td><td></td></tr><tr><td>PVDF membrane</td><td>Vayzme</td><td>E801</td><td></td></tr><tr><td>SDS powder</td><td>Solarbio</td><td>S8010-500g</td><td></td></tr><tr><td>SDS-PAGE sysytem</td><td>Aervicebio</td><td>BVE4-BVT4</td><td></td></tr><tr><td>Sonics</td><td>Uibra cell</td><td></td><td></td></tr><tr><td>Tris-HCl powder</td><td>Biotopped</td><td>HST-0372</td><td></td></tr><tr><td>Triton X-100</td><td>Servicebio</td><td>GC204003-100ml</td><td></td></tr><tr><td>Trypsin</td><td>Servicebio</td><td>G4021-100ML</td><td></td></tr><tr><td>Vortex mixer</td><td>Kindly donated by Vayzme</td><td></td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Aladdin</td><td>M301573</td><td></td></tr></tbody></table>

comparative strategies for ubiquitination detection in mammalian cell lysates using smad2/smurf2 as a model

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions by promoting proteasomal degradation or altering subcellular localization through ubiquitin chain-dependent signaling. Here, we describe two cell lysis methods for detecting SMAD2 ubiquitination levels in HEK293T cells and compare their effectiveness in analyzing protein ubiquitination levels. Protein overexpression in cells was induced by transient transfection. The plasmids (HA-Ub, FLAG-SMAD2, and MYC-SMURF2) and transfection reagents were separately added to basal medium, mixed, and the mixture was added to the cells. Prior to harvest, MG132 was added to inhibit proteasomal degradation and enhance ubiquitinated protein accumulation. The primary divergence between the two experimental approaches is their cell lysis methods-the ice-bath method (performed at 4 &#176;C) and the heat-treatment method (involving incubation at 95 &#176;C), which substantially affects the efficiency of protein lysis. After cell lysis was completed, the cell lysate, agarose beads, and FLAG antibody were mixed and incubated at 4 &#176;C overnight. Ubiquitinated proteins were then detected by western blot analysis. Before detecting ubiquitinated proteins, a light-chain antibody was used for secondary antibody incubation. Then, ubiquitination bands were detected. The results show that both the ice-bath method and the heat-treatment method can be used to detect ubiquitination levels, while the heat-treatment method may make it easier to detect ubiquitination of SMAD2. This study delineates and compares two cell lysis methods for measuring ubiquitination levels in mammalian cells, using SMURF2/SMAD2 as a model, to assist researchers in selecting more appropriate methods for detecting the ubiquitination levels of substrate proteins.

As a "molecular messenger" in TGF-β signaling pathway, SMAD2, after being phosphorylated byTGF-β receptors, forms a complex with SMAD3 and SMAD4, translocates to the nucleus, and initiates the transcription of downstream target genes30. It is thus a key molecule linking extracellular signals to intracellular gene expression. Its degradation is mediated by SMURF2, an E3 ubiquitin ligase that catalyzes the polyubiquitination of the target protein, thereby targeting it for proteasomal degradation. Th...

Ubiquitination is a critical regulator of intracellular protein functions. Here, we describe two cell lysis methods for detecting substrate protein ubiquitination levels in mammalian cells and compare their effectiveness in analyzing ubiquitination levels. Protein overexpression in HEK293T cells was induced by transient transfection, and ubiquitinated proteins were detected by western blot analysis.

Ubiquitination, a post-translational modification, is a critical regulator of intracellular protein function. Ubiquitination modulates protein functions ...

Ubiquitination governed by the E one, E two, E three enzymatic cascade is a key regulator of protein function. Ubiquitin structural flexibility allows it to form diverse chains via different lysine or Met one linkages with each topology dictating a distinct outcome, K 48 for degradation, K 63 for signaling and repair and Met one for innate immunity. This study delineates and compares two different cell lysis methods to measure application levels in the mammalian cells using Smurf two, SMAD two as a model, which will assist researchers in selecting more appropriate methods to detect the ubiquitination level of substrate proteins.Maintain HEK two 93 T cells in high-glucose DMEM supplemented with 10%FBS and one percent PS.Seed cells in six well plates at 500, 000 cells per well. Prepare transfection mixtures to represent 10%of the total culture value and use SMAD two ubiquitination as a experimental model. Combine different plasmids in 100 microliter of serum-free DMEM.In a separate tube, dilute transfection reagent. As the reagent mixture dropwise to the plasmid solution, incubate for 15 minutes at room temperature and distribute evenly onto cells. Replace the transfection medium after six to hours with two microliter of fresh complete DMEM per well.Treat cells with five nanometer MG one three two for 12 hours before harvesting. Aspirate medium and wash each well with one mil of prechilled PBS. Remove PBS completely.Transfer the suspension to a one point five tube and place on ice. Centrifuge at three hundred and 80 XG for five minutes at four degrees. Remove the supernatant and retain the pellet.Transfer 50 microliter of protein AG agarose beads per sample to a new tube. Wash beads three times with one mil of lysis buffer. Remove residual buffer without disturbing the pellet.At 100 microliter of working solution and five microliter of antibody. Add 500 microliter of prechilled working solution to the pellet. Incubate on ice for 30 minutes.Sonicate at two W using four secs on, four sec off cycles for five repetitions. Return samples to ice for the remainder of the incubation. Add 40 microliter of working solution to the pellet.Add five microliter over 10%SDS while mixing continuously. Vortex briefly and heat at 95 degrees until the suspension becomes transparent and fully liquified. Invert intermittently to ensure uniform denaturation.Add 450 microliter working solution. Sonicate at two W using four secs on four secs off cycles for five repetitions. Centrifuge lysates for 15 minutes at four degrees.Reserve 60 microliter of lysate and add 15 microliter of five multi loading buffer. Heat at 95 degrees for 15 minutes and label as input. Add 100 microliter of antibody-coupled beads to the remaining lysate.Incubate at four degrees for eight to 12 hours with rotation. Centrifuge beads at 100 XG for one minutes at four degrees and discard supernatant. Add 75 microliter of one multi SDS loading buffer and hit at 95 degrees for 15 minutes to elute proteins, label as IP.Detect ubiquinated proteins by western blot.HE K two 93 T cells were transfected and stimulated with MG one 32. The cells were divided into five groups according to different treatment methods and the differences between each group are as follows, without flag SMAD two lane one, without MG one three two lane two, without FLAG antibody added into beads lane three, with MYC-SMURF two lane five. All cells were lysed using the ice-bath method.Lane one served as a control to verify the successful over expression of the SMAD two plasmid. Lane two served as a control to demonstrated the inhibitory effect of MG one three two on the degradation of ubiquitinated proteins. Lane three served as a control to confirm that nonspecific binding has been completely eliminated.Lane five was used to investigate the promoting effect of SMURF two on the ubiquitination of SMAD two. The greyscale value of HA in lane five was higher than that in lane four, which demonstrated that the SMURF two increased the ubiquitination level of SMAD two. When we lysecells using the heat treatment method we obtained the similar results as those with the ice-bath method, however, when using the heat treatment method, the expression of SMURF two could not be detected in the IP FLAG results.We also demonstrated that the heat treatment method is also suitable for the detection of ubiquitination of endogenous proteins. We further compare the efficiency of the two methods in detecting ubiquitination levels whether in the input or IP flag results we found that the greyscale value of HA in the group using the heat treatment method was higher than that in the ice-bath method. To enhance signaling detection, we employed plasmid based over expression combined with MG one three two treatment during cell harvesting to prevent degradation of ubiquitinated proteins.Lysate preparation requires careful modulation of na cl concentration as high salt disrupts weak interaction while low salt increases nonspecific aggregation. However, low concentrations may lead to excessive background from nonspecific aggregates. For enhanced protein enrichment recovery antibodies to beads at four degrees ensure more efficient capture of target proteins and reduces non-specific binding.In heat treatment method, it's critical to ensure that concentration of SDS remains below one percent, access SDS may strip antibodies from beads. This study delineates and comparisons to different lysis methods to measure ubiquitination levels in mammalian cells using SMURF two SMAD two as a model which will assist researchers in selecting more appropriate methods to detect the ubiquitination levels of substrate proteins.

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Research

JoVE Journal

Biology

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.

Detection of Protein Ubiquitination

This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting technique.

SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

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.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

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.

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

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.

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

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.

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In Vitro Analysis of E3 Ubiquitin Ligase Function

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.

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

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.

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Learn two cell lysis methods for detecting ubiquitination in mammalian cells using SMAD2/SMURF2 as a model. This protocol compares ice-bath and heat treatment approaches to help researchers select optimal techniques.

Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model

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