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Ubiquitinated Proteins

Ubiquitinated Proteins: Discover the Intricacies of Protein Modification

Ubiquitinated proteins are a critical component of cellular regulation, playing crucial roles in protein degradation, signaling pathways, and disease processes.
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Most cited protocols related to «Ubiquitinated Proteins»

To functionally annotate proteins regulated by ubiquitination, we downloaded a set of 5,884 verified ORFs (5817 sequences of length ≥50) from the SGD website and applied UbPred. A major challenge in finding proteins that are most likely to be ubiquitinated is a possibility that a direct application of UbPred to any proteome would favor longer proteins, as a consequence of <100% prediction accuracy. Thus, to extract a set of proteins with strongest predictions, we proceeded as follows.
First, a threshold t was determined such that only 100·p% of all prediction scores over all proteins were greater than t. For a sufficiently high t, or similarly, sufficiently low p, such scores can be considered as strong predictions of ubiquitination, which is supported by the low false positive rate in the bottom left-hand corner of the estimated ROC curve. Then, with a reasonable assumption, we introduced a null model in which a randomly selected lysine from any protein had 100·p% chance of being predicted as strong. Under this model, the number of strong predictions (with scores above threshold t) in each protein would be proportional to the number of lysines it contains. Therefore, using the null model assumption, the probability that, in a protein containing K lysines, the number of strong predictions that occurred by chance is k or greater, can be expressed as
P=i=kK(Ki)·pi·(1p)Ki where p is the probability that a randomly selected lysine has a strong prediction of being ubiquitinated. Thus, proteins with the lowest P-value P are the most likely to contain a disproportionately larger number of strong predictions than expected by chance. We considered these proteins to be the most strongly ubiquitinated proteins (i.e. over-ubiquitinated). The potential length dependence was thus eliminated since the P-values implicitly equalize the length factor. We selected the threshold of p = 0.1 and extracted all proteins with P < 0.05, Bonferroni corrected. In addition, since consecutive lysines may not be considered to be motionally independent (possibly invalidating null model assumptions), we note that a selection of the smaller samples of lysines from each protein did not significantly influence the results reported herein.
Publication 2009
Lysine Open Reading Frames Proteins Proteome SET protein, human Staphylococcal Protein A T protein, human Ubiquitinated Proteins Ubiquitination
For IP and western blot, dynabeads m-270 Epoxy (Invitrogen) coupled with antibodies were prepared and then cell lysates were added, and the antibodies−lysate mixtures were rotated at 4 °C for 1 h. Immunocomplexes separated from the dynabeads were washed with lysis buffer and then suspended with SDS blue loading buffer. To detect ubiquitinated proteins, lysis was performed under 80 °C for 10 min. Western blot analysis was performed as we described previously.40 (link) In brief, an equal amount of total protein extracted from cultured cells were separated by 12% SDS–PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked with 5% milk for 1 h. Primary Abs and horseradish peroxidase-conjugated secondary Abs were each incubated for 1 h. The bounded secondary antibodies were reacted to the ECL detection reagents and exposed to X-ray films (Kodak, Japan).
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Publication 2017
Antibodies Buffers Cells Cultured Cells Epoxy Resins Horseradish Peroxidase Milk, Cow's polyvinylidene fluoride Proteins SDS-PAGE Tissue, Membrane Ubiquitinated Proteins Western Blot Western Blotting X-Ray Film

Arabidopsis leaves were collected before and after heat treatment, ground in liquid nitrogen and homogenized in an detergemt-containing extraction buffer (100 mMTris/HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2% ß-mercaptoethanol). The homogenates were filtered through a 300 µm and 100 µm nylon mesh and clarified by centrifugation at 2,200xg for 5 minutes. Supernatants were kept for further analysis. The pellets were resuspended in the same buffer and subjected to the low speed centrifugation. The process was repeated twice and after the last centrifugation the pellets were resuspended in the extraction buffer. The concentrations of proteins in the homogenates (total proteins), the first supernatants (soluble proteins) and last pellets (insoluble proteins) were determined using Bio-Rad protein assay kit. The first supernatant fractions and last pellets were separated by SDS–PAGE. For western blotting, fractionated proteins on SDS/PAGE gel were transferred to nitrocellulose membrane. NBR1-TAP was detected by a peroxidase-conjugated anti-peroxidase antibody. Ubiquitinated proteins were detected by protein blotting using an anti-ubiquitin monoclonal antibody (Sigma, USA). The antigen-antibody complexes were detected by enhanced chemiluminescence using luminal as substrate as previously described [39] .
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Publication 2013
2-Mercaptoethanol Antibodies, Anti-Idiotypic Arabidopsis Biological Assay Buffers Centrifugation Chemiluminescence Complex, Immune Edetic Acid Monoclonal Antibodies Nitrocellulose Nitrogen Nylons Pellets, Drug Peroxidase Phenobarbital Proteins RRAD protein, human SDS-PAGE Sodium Chloride Tissue, Membrane Triton X-100 Ubiquitin Ubiquitinated Proteins
Tissue samples were processed for Western blotting using our standard method, as described.7 Briefly, tissue samples (brain cortex, spinal cord, and kidney) were dissected on ice and snap frozen in liquid nitrogen as quickly as possible to prevent deconjugation of SUMOylated and ubiquitinated proteins. Tissue samples were homogenized by sonication using lysis buffer supplemented with 2% SDS. Quantification of signal intensities was performed using ImageJ (NIH, Bethesda, MD). The primary antibodies are listed in Table 2.
Publication 2018
Antibodies Brain Buffers Cortex, Cerebral Freezing Kidney Nitrogen Spinal Cord Tissues Ubiquitinated Proteins

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Publication 2012
alpha-Synuclein Brain Buffers Clone Cells Complex, Immune Electrophoresis G-substrate Immunoglobulins Immunoprecipitation Mice, House Monoclonal Antibodies Phosphoric Monoester Hydrolases Polyubiquitin polyvinylidene fluoride Protease Inhibitors Proteins Radioimmunoprecipitation Assay Technique, Dilution Temporal Lobe Tissue, Membrane Ubiquitin Ubiquitinated Proteins Western Blotting

Most recents protocols related to «Ubiquitinated Proteins»

Ubiquitinated NKCC2 was measured as previously described (7 (link)). Briefly, ubiquitinated proteins were isolated using a Ubiqapture-Q kit (Enzo Life Sciences, Farmingdale, NY) following the manufacturer’s instructions. To measure ubiquitinated NKCC2 and the effect of cGMP, the whole TAL sample was treated with the proteasomal inhibitor MG132 (20 µM). TALs were split and treated with vehicle or inhibitor for 10 min at 37°C. Vehicle or dybutyryl cGMP (db-cGMP) was then added to the respective samples and incubated for 50 min at 37°C. Once the treatment was finalized, samples were cooled with chilled PS. Suspensions were centrifuged at 120 g for 2 min at 4°C. The PS was discarded, and TALs were incubated with lysis buffer [containing 150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 2% Triton X-100, and 0.2% SDS and supplemented with protease inhibitors, namely, 10 µg/mL aprotinin, 5 µg/mL leupeptin, 4 mmol/L benzamidine, 5 µg/mL chymostatin, and 5 µg/mL pepstatin-A; pH 7.5 (Sigma)]. TALs were vigorously vortex three times for 3 s each. Each tube was spun at 12,000 g for 2 min at 4°C. The undissolved pellet was discarded. The protein content in each sample was measured in duplicate with a colorimetric assay using Bradford’s method (Pierce Biotechnology, Rockford, IL). The TAL lysate (150 μg protein) was incubated on a rocking platform at 4°C overnight with 40 μL of a 50% slurry containing Ubiqapture-Q beads in a final volume of 400 μL. The beads were centrifuged at 12,000 g for 2 min at 4°C. The supernatant was separated from the beads and saved for later measurement of nonubiquitinated NKCC2 and was also used as a loading control. The beads were washed twice with high-salt buffer (500 mM NaCl and 50 mM HEPES; pH 7.4) and twice with no-salt buffer (50 mM HEPES; pH 7.4). Proteins were eluted from the beads by boiling in 60 μL of SDS Laemmli loading buffer containing 50 μM dl-dithiothreitol and 5% β-mercaptoethanol. Proteins from the supernatant and proteins eluted from the beads were separated by SDS-PAGE (6% gels) and transferred to Immobilon PVDF membranes (Millipore, Bedford, MA). NKCC2 was detected by Western blot analysis.
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Publication 2023
2-Mercaptoethanol Aprotinin benzamidine Biological Assay Buffers chymostatin Colorimetry Cyclic GMP Dithiothreitol Edetic Acid Gels HEPES Immobilon Laemmli buffer leupeptin MG 132 Microcephalic Osteodysplastic Primordial Dwarfism, Type I pepstatin polyvinylidene fluoride Protease Inhibitors Proteasome Inhibitor Proteins SDS-PAGE Sodium Chloride Tissue, Membrane Triton X-100 Ubiquitinated Proteins Western Blot
Immunoprecipitation assays were carried out as described previously [62 (link)]. The indicated cell lysates were hatched with specific primary antibodies and Protein A + G magnetic beads (MedChemExpress, Monmouth Junction, NJ, USA) overnight at 4 °C. Finally, after being washed with a lysis buffer, the immunocomplexes were analyzed via immunoblotting using the corresponding antibodies.
For ubiquitination analysis, cells were transfected with the specified plasmid and treated with MG132 (MedChemExpress) before treatment with lysis buffer. Immunoprecipitation was performed on cell lysates using the indicated primary antibodies. Then, the ubiquitinated proteins were hatched with protein A/G magnetic beads and detected via immunoblotting using specific primary antibodies.
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Publication 2023
Antibodies Biological Assay Buffers Cells G-substrate GTP-Binding Proteins Immunoprecipitation MG 132 Plasmids Staphylococcal Protein A Ubiquitinated Proteins Ubiquitination
Ubiquitination assay was carried out in HEK293T cells. In HEK293T cells, HA-Ub and Flag-YAP1 were co-transfected with empty vector, Myc-PDLIM7 or Myc-TPM2 plasmids. Cells were treated with 10 mM MG132 for 6 h before harvest. Ubiquitinated YAP1 proteins were enriched by anti-Flag magnetic beads. After eluted with the Flag peptide, ubiquitinated YAP1 was detected by anti-HA antibody.
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Publication 2023
Antibodies, Anti-Idiotypic Biological Assay Cells Cloning Vectors FLAG peptide MG 132 Plasmids Ubiquitinated Proteins Ubiquitination YAP1 protein, human
The Signal-Seeker Ubiquitin Enrichment Kit (BK161, Cytoskeleton) was used according to the manufacturer’s instructions to pull down ubiquitinated proteins. Briefly, H1975 cells with or without MTSS1 overexpression were collected and lysed with BlastR™ lysis buffer with inhibitors (de-ubiquitinase inhibitor, Cat # NEM09BB; protease inhibitor cocktail, Cat # PIC02). The lysates were transferred into BlastR™ filters and diluted with BlastR™ dilution buffer to the final volume. Equal amounts of protein were incubated with 20 µL ubiquitination affinity beads or control beads for 2 h at 4 °C. The beads were washed 3 times with BlastR-2™ wash buffer, followed by incubation with 30 µL elution buffer for 5 min at room temperature. The precipitates were collected by the spin columns provided in the kit and were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For His-tagged ubiquitin pulldown with Ni–NTA beads, HeLa cells were transfected with wild-type (His–Ubiquitin–WT) or lysine-mutated (His–Ubiquitin–KO) ubiquitin and the other plasmids for 60–72 h. Cells were harvested and resuspended in Buffer A (6 M guanidine–HCl, 0.1 M Na2HPO4/NaH2PO4, 10 mM imidazole pH 8.0) with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The lysates were sonicated (75 W, 2 s, 5 s, 3 min) before mixing with Ni–NTA beads (QIAGEN) by rotating at room temperature for 3 h. Subsequently, the His pull-down products were washed twice with buffer A, twice with buffer A/TI (1 volume buffer A and 3 volumes buffer TI), and once with buffer TI (25 mM Tris-HCl and 20 mM imidazole, pH 6.8). Then elution buffer (0.2 M imidazole, 5% w/v SDS, 0.15 M Tris-Cl, pH 6.8) was added and incubated for 20 min at room temperature. The supernatants were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
For Flag-tagged ubiquitin pulldown, 293 T or HeLa cells transfected with Flag-ubiquitin were lysed with IP buffer with inhibitors (10 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 5 mM N-Ethylmaleimide and Protease inhibitor cocktail). The subsequent immunoprecipitation was performed as described above. For PD-L1 deglycosylation, immunoprecipitates or whole cell lysates were treated using PNGase F according to the manufacturer’s instructions. Finally, samples were boiled for 10 min at 95 °C before being resolved by SDS-PAGE and immunoblotted.
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Publication 2023
Buffers CD274 protein, human Cells Cytoskeleton Ethylmaleimide Glycopeptidase F Guanidine HeLa Cells imidazole Immunoprecipitation inhibitors Lanugo Lysine Plasmids Protease Inhibitors Proteins SDS-PAGE Technique, Dilution Tromethamine Ubiquitin Ubiquitinated Proteins Ubiquitination ubiquitin isopeptidase
A total of 80,000 cultured DRG neurons per treatment (40,000 neurons/well and two wells were combined for each experiment) were infected with HSV-1 KOS or HSV-1 n212, or were uninfected, and treated with MG132 (Cbz-Leu-Leu-Leucinal) to inhibit the ubiquitin-proteasomal degradation complex and preserve ubiquitinated proteins post-inoculation. The infection progressed for 8 h prior to protein harvesting in 125 μL non-denaturing lysis buffer (20 mM Tris HCl pH8, 1% NP-40, 2 mM EDTA) with MG132, PR619 (2,6-Diaminopyridine-3,5-bis(thiocyanate)) broad-spectrum deubiquitinating enzyme inhibitor to prevent the removal of ubiquitin moieties after collection, and Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Samples were incubated for 12 h with FK2 anti-ubiquitin antibody covalently conjugated to Invitrogen Dynabeads (Thermo Fisher Scientific), according to the manufacturer’s protocol. Immunoprecipitated samples were rinsed 3 times with non-denaturing buffer and resuspended in non-denaturing lysis buffer with MG132, PR619, and Halt Protease & Phosphatase Inhibitor. Samples were shipped overnight to MSBioworks (Ann Arbor, MI, USA) for LC-MS/MS analysis. Each sample was eluted in 70 μL 1.5× NuPage LDS Sample Buffer (Thermo Fisher Scientific) and boiled at 100 °C for 15 min, followed by clarification via centrifugation. Half of each sample was processed by SDS-PAGE using a 10% Bis-Tris NuPage Mini-gel (Thermo Fisher Scientific) with an MES buffer system. A 2 cm gel space was excised into ten bands, washed with 25 mM ammonium bicarbonate and acetonitrile, reduced with 10 mM dithiothreitol at 60 °C, alkylated with 50 mM iodoacetamide at room temperature, digested with trypsin at 37 °C for 4 h, quenched with formic acid, and finally analyzed using a nano LC-MS/MS with Waters M-Class LC system interfaced to a Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Each sample was analyzed for 5 h. The infection was repeated a second time, using neuronal cultures performed on a different day and following the identical processes and protocols to generate a replicate set of mass spectrometry data.
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Publication 2023
acetonitrile ammonium bicarbonate Antibodies, Anti-Idiotypic Bistris Buffers Cardiac Arrest Centrifugation Deubiquitinating Enzymes Dithiothreitol DNA Replication Edetic Acid Enzyme Inhibitors formic acid Human Herpesvirus 1 Infection Iodoacetamide leucinal leucylleucine Mass Spectrometry MG 132 Multicatalytic Endopeptidase Complex Neurons Nonidet P-40 Phosphoric Monoester Hydrolases Protease Inhibitors Proteins SDS-PAGE Tandem Mass Spectrometry Thiocyanates Tromethamine Trypsin Ubiquitin Ubiquitinated Proteins Vaccination

Top products related to «Ubiquitinated Proteins»

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MG132 is a proteasome inhibitor, a type of laboratory reagent used in research applications. It functions by blocking the activity of the proteasome, a complex of enzymes responsible for the degradation of proteins within cells. MG132 is commonly used in cell biology and biochemistry studies to investigate the role of the proteasome in various cellular processes.
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N-ethylmaleimide is a chemical compound used in various laboratory applications. It functions as a reagent for the selective modification of sulfhydryl groups in proteins and peptides.
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Ni-NTA agarose beads are a chromatography resin used for the purification of His-tagged proteins. The beads consist of a nickel-nitrilotriacetic acid (Ni-NTA) complex immobilized on an agarose matrix. These beads can selectively bind and capture proteins with a polyhistidine (His-tag) affinity tag, allowing for their efficient separation and purification from complex mixtures.
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Ubiquitin is a small, regulatory protein found in eukaryotic cells. Its primary function is to tag other proteins for degradation by the proteasome, a complex that breaks down unwanted or damaged proteins within the cell.
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Ni-NTA beads are a type of agarose-based affinity resin used for the purification of recombinant proteins that contain a polyhistidine (His) tag. The Ni-NTA (Nickel-Nitrilotriacetic Acid) moiety on the beads binds to the His-tagged proteins, allowing them to be separated from other cellular components during the purification process.
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Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
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Ni2+-NTA agarose beads are a type of affinity chromatography resin used for the purification of histidine-tagged proteins. The beads consist of nickel-nitrilotriacetic acid (Ni2+-NTA) covalently attached to an agarose matrix. The Ni2+ ions on the beads bind to the histidine tags on the target proteins, allowing their capture and subsequent elution.
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The Anti-FLAG M2 affinity gel is a chromatography resin designed for the purification of recombinant proteins tagged with the FLAG peptide sequence. The gel consists of an agarose matrix covalently coupled with the anti-FLAG M2 monoclonal antibody, which specifically binds to the FLAG tag. This allows for the selective capture and purification of FLAG-tagged proteins from complex mixtures.
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More about "Ubiquitinated Proteins"

Ubiquitinated proteins are a critical component of cellular regulation, playing pivotal roles in protein degradation, signaling pathways, and disease processes.
These modified proteins, also known as ubiquitin-conjugated proteins, are regulated by the ubiquitin-proteasome system (UPS), a complex machinery responsible for the targeted destruction of unwanted or damaged proteins.
The process of ubiquitination involves the covalent attachment of the small, regulatory protein ubiquitin to specific lysine residues on target proteins.
This tagging system marks the proteins for degradation by the 26S proteasome, a multi-subunit complex that breaks down the ubiquitinated proteins into smaller peptides.
Researchers can leverage various techniques and tools to study ubiquitinated proteins, such as the use of the proteasome inhibitor MG132, which blocks the degradation of ubiquitinated proteins, and N-ethylmaleimide, which prevents the deubiquitination of proteins.
Affinity-based purification methods, like those employing Ni-NTA agarose beads or Anti-FLAG M2 affinity gel, can be used to isolate and identify ubiquitinated proteins.
Ubiquitin, the key player in this process, is a small, highly conserved protein that can be conjugated to target proteins, marking them for degradation.
The Ubiquilin 1 Tandem UBA (TUBE2) Agarose is a useful tool for the enrichment and study of ubiquitinated proteins.
Understanding the complex mechanisms of ubiquitination and the interplay of ubiquitinated proteins is crucial for unraveling the intricacies of cellular regulation and disease pathogenesis.
By utilizing the powerful AI-driven platform of PubCompare.ai, researchers can optimize their research protocols, identify the best products and procedures, and achieve enhanced reproducibility and accuracy in their studies on ubiquitinated proteins.