Anti ubiquitin

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-ubiquitin is a lab equipment product that detects and binds to ubiquitin, a small regulatory protein found in all eukaryotic cells. It is used to identify and study ubiquitinated proteins in various biological samples.

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Lab products found in correlation

Close spelling variants of this product

Ubiquitin (177 citations) Anti-ubiquitin antibody (41 citations) Mouse anti-ubiquitin (18 citations) Anti-ubiquitin (P4D1) (14 citations) Anti-ubiquitin (3936) (9 citations) Rabbit anti-ubiquitin (9 citations) Anti-K48 ubiquitin (8 citations) Anti-ubiquitin #3933 (5 citations) Anti-K48-linked ubiquitin (3 citations) Anti‐ubiquitin Ab (3 citations)

116 protocols using anti ubiquitin

Antibody Validation for SMPD3 and AMPK

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Rabbit polyclonal antibody against phosphor-SMPD3-S209/208 (ABclonal, cat. #AP1312, 1:500 for IB) was raised and validated by Abclonal. Rabbit polyclonal antibody against ubiquitin-SMPD3-K103 (ABclonal, cat. #A21272, 1:500 for IB) was raised and validated by Abclonal. Rabbit anti-phospho-AMPKα-T172 (Cell Signaling Technology, cat. #2535, 1:1000 for IB), anti-AMPKα (Cell Signaling Technology, cat. #2532, 1:1000 for IB), anti-AMPKα1 (Cell Signaling Technology, cat. #2795, 1:1000 for IB) anti-AMPKα2 (Cell Signaling Technology, cat. #2757, 1:500 for IB), anti-ACTB (ABclonal, cat. #ab8227, 1:2000 for IB), anti-SMPD3 (Santa Cruz Biotechnology, cat. #sc-166637, 1:500 for IB), Anti-Ubiquitin (Medical & Biological Laboratories, cat. #MK-11-3, 1:500 for IB), Anti-Ubiquitin (linkage-specific K48) (Cell Signaling Technology, cat. #8081, 1:500 for IB), Anti-Ubiquitin (linkage-specific K63) (Cell Signaling Technology, cat. #5621, 1:500 for IB) were used in this study.

Chen B., Sun L., Zeng G., Shen Z., Wang K., Yin L., Xu F., Wang P., Ding Y., Nie Q., Wu Q., Zhang Z., Xia J., Lin J., Luo Y., Cai J., Krausz K.W., Zheng R., Xue Y., Zheng M.H., Li Y., Yu C., Gonzalez F.J, & Jiang C. (2022). Gut bacteria alleviate smoking-related NASH by degrading gut nicotine. Nature, 610(7932), 562-568.

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Quantification of α-Synuclein Aggregates in Neurons

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Neurons were fixed with 4% paraformaldehyde (PFA) and 0.1% TX‐100 in PBS for 15 min. To detect the insoluble α‐syn aggregates, α‐syn‐HEK293 cells were fixed with 4% PFA in PBS followed by permeabilization with 1% TX‐100. Cells were incubated with anti‐pS129 (1:1000, Biolegend, 825701), anti‐Ubiquitin (1:500, Cell Signaling Technology, #3936) overnight at 4°C. The samples were stained with corresponding secondary antibodies Alexa Fluor 594 or 488 (1:1000, Invitrogen). Nuclei were visualized with DAPI (1 μg/ml, BioFroxx, 1155MG010) for 5 min. To quantify the percentage of positive cells, a total of 8 fields, each with 100+ cells, were analyzed per condition. The primary cultured neurons and brain sections were double stained for MAP2/pS129, pS129/K80Hcy, Thioflavin S (ThS)/K80Hcy, TH/pS129, TH/K80Hcy, or GFP/TH.

Zhou L., Guo T., Meng L., Zhang X., Tian Y., Dai L., Niu X., Li Y., Liu C., Chen G., Liu C., Ke W., Zhang Z., Bao A, & Zhang Z. (2022). N‐homocysteinylation of α‐synuclein promotes its aggregation and neurotoxicity. Aging Cell, 22(3), e13745.

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Detergent-Soluble and Insoluble Fractions Analysis

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Western blots for detergent-soluble and insoluble fractions were done as before.^{20 (link),32 (link),86 (link)} In brief, thoraces were dissected from 20 male flies/sample and homogenized in ice-cold PBS with 1% Triton X-100 containing protease and phosphatase inhibitors. Homogenates were centrifuged at 14,000 rpm at 4°C and supernatants collected (Triton X-100 soluble fraction). The remaining pellet was washed in ice-cold PBS with 1% Triton X-100. The pellet was then resuspended in RIPA buffer containing 8M urea and 5% SDS, centrifuged at 14,000 rpm at 4°C. The supernatants (Triton X-100 insoluble fraction) were collected and analyzed on 4–20% SDSPAGE with anti-ubiquitin (Cell Signaling Technologies P4D1, #3936) and anti-Ref(2)P/p62 (Abcam #178840) antibodies.

Jiao J., Curley M., Graca F.A., Robles-Murguia M., Shirinifard A., Finkelstein D., Xu B., Fan Y, & Demontis F. (2023). Modulation of protease expression by the transcription factor Ptx1/PITX regulates protein quality control during aging. Cell reports, 42(1), 111970.

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Western Blot Analysis of Cell Signaling

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Western blot analysis was performed according to a standard protocol.⁴⁴ (link) The primary antibodies included anti-ACP5 (Gentex, CA, USA, 1:1,000), anti-fibronectin, anti-E-cadherin, anti-vimentin (Proteintech, Wuhan, China, 1:1,000), anti-p38, anti-ERK, anti-p-ERK, anti-AKT, anti-β-catenin, anti-SMAD2/3, anti-p-SMAD2, anti-p-SMAD3, anti-p53, anti-p53 (Ser392), anti-ubiquitin (Cell Signaling Technology, Danvers, MA, USA, 1:1,000), anti-GAPDH, and anti-β-actin (Abcam, Cambridge, MA, USA, 1:3,000) antibodies. Detection was performed using a chemiluminescent substrate system (Bio-Rad, Hercules, CA, USA). The gray values were analyzed with ImageJ software.

Hu Y., Yu J., Wang Q., Zhang L., Chen X., Cao Y., Zhao J., Xu Y., Jiang D., Wang Y, & Xiong W. (2020). Tartrate-Resistant Acid Phosphatase 5/ACP5 Interacts with p53 to Control the Expression of SMAD3 in Lung Adenocarcinoma. Molecular Therapy Oncolytics, 16, 272-288.

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Protein Expression Analysis by Western Blot

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Cells were lysed on ice in buffer containing 50mM HEPES, pH7.4, 80mM NaCl, 5mM MgCl₂, 10mM EDTA, 5mM sodium pyrophosphate*10H₂O, 1% TritonX-100, and Protease Inhibitor Cocktail (Sigma-Aldrich). 30 μg of total protein from each sample was resolved on Novex 4-20% Tris-Glycine Mini Protein Gels and transferred onto nitrocellulose membranes. The blots were probed with the appropriate antibodies: anti-ubiquitin (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), anti-20S/β1 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β2 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β5 subunit (Santa Cruz Biotechnology, Inc.), anti-E-cadherin (BD Biosciences), anti-fibronectin (BD Biosciences), anti-vimentin (Cell Signaling Technology), anti-phospho-Smad2 (Ser465/467) (Cell Signaling Technology), or anti-Smad2/3 (Cell Signaling Technology). Signals were detected using Odyssey infrared imaging system (LI-Cor Biosciences).

Banno A., Garcia D.A., van Baarsel E.D., Metz P.J., Fisch K., Widjaja C.E., Kim S.H., Lopez J., Chang A.N., Geurink P.P., Florea B.I., Overkleeft H.S., Ovaa H., Bui J.D., Yang J, & Chang J.T. (2016). Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition. Oncotarget, 7(16), 21527-21541.

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Subcellular Protein Extraction and Immunoblotting

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All cells were briefly rinsed with cold PBS prior to collection. Both whole cells and subcellular fractions were lysed in RIPA buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 0.5% SDS, 0.5% sarkosyl, 0.5% NP40, 20 mM EDTA, Roche protease inhibitors, 1 mM PMSF) on ice. Protein concentrations of lysates were determined by the bicinchonic acid assay (Thermo Fisher) before being analyzed by SDS-PAGE. The primary antibodies used included anti-CARM1 (Cell Signaling Technology, 3379), anti-skp2 (Santa Cruz Biotechnology, sc-7164), anti-LC3B (Cell Signaling Technology, 3868), anti-LAMP1 (Cell Signaling Technology, 3243), anti-C9orf72 (Bio-Rad VMA00065), anti-PARP (Cell Signaling Technology, 9542), anti-β-actin (Santa Cruz Biotechnology, sc-47778), anti-NOX2 (Abcam, ab129068), anti-ubiquitin (Cell Signaling Technology, 3936), anti-calnexin (Cell Signaling Technology, 2679P), anti-ACC (Cell Signaling Technology, 3676), and anti-ADRP (Progen Biotechnik, GP42).

Liu Y., Wang T., Ji Y.J., Johnson K., Liu H., Johnson K., Bailey S., Suk Y., Lu Y.N., Liu M, & Wang J. (2018). A C9orf72–CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress. Genes & Development, 32(21-22), 1380-1397.

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Lysis Conditions for Preserving Ubiquitin Conjugations

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Cells were washed with PBS and then lysed with either the lysis buffer (#9803; Cell Signaling Technology; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na₃VO₄, 1 μg/mL leupeptin) or the acidic lysis buffer (50 mM HEPES, pH 6.0, 150 mM NaCl, 0.1% (w/v) SDS) supplemented with protease cocktail (Roche). The acidic lysis buffer was used to preserve the ubiquitin or ubiquitin-like conjugations (e.g., UBA3^N8, Ubc12^N8, UbcH7^Ub, Ubc9^SUMO) that are labile in the regular lysis buffer. The supernatant of the lysate was used for Western blotting. The antibodies used in this study include anti-cullin1 (H213; Santa cruz), anti-UBA3 (F-10; Santa cruz), anti-GlyRS (B01P; Abnova or sc-98614; Santa cruz), anti-SerRS (homemade), anti-V5 (R96-CUS; Invitrogen), and anti-Ube2F (pa5-26641; Thermo Fisher). Other antibodies including anti-NEDD8 (#2754), anti-Ubiquitin (#3936), anti-SUMO1 (#4930), anti-Ubc12 (#5641), anti-Ubc9 (#4918), anti-Flag (#2908), anti-UBA1(#4891), anti-UbcH7 (#3848), anti-UBA2 (#8688), anti-APPBP1 (#14321), anti-p27kip (#3698), and anti-α-Tubulin (#3873) are from Cell Signaling.

Mo Z., Zhang Q., Liu Z., Lauer J., Shi Y., Sun L., Griffin P.R, & Yang X.L. (2016). Neddylation requires glycyl-tRNA synthetase to protect activated E2. Nature structural & molecular biology, 23(8), 730-737.

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Inflammatory Signaling Pathway Analysis

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Carnosic acid, 5-aminosalicylic acid (5-ASA), sodium carboxymethyl cellulose, haematoxylin and eosin were purchased from Sigma Aldrich (St. Louis, MO). Dextran sodium sulfate was purchased from MP Biomedicals (Solon, OH). Anti-p65 (#8242), anti-phospho-p65 (#3039), anti-IĸBα (#9242), anti-phospho-IĸBα(#2859), anti-Stat3 (#4904), anti-phospho-Stat3 (#9145), anti-JNK (#9252), anti-phospho-JNK (#4668), anti-c-Jun (#2315), anti-phospho-c-Jun (#2361), anti-NLRP3 (#15101), anti-ASC (#67824), anti-Ubiquitin (#3933), iNOS (#13120), anti-H3k27Me3 (#9733), anti-H3k4Me3 (#9751) and anti-β-actin (#4970) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Keap1 (ab150654), anti-Nrf2 (ab62352) and Cullin3 (ab75851) antibodies were purchased from ABCAM. Anti-caspase1 (sc-56036) antibody was purchased from Santa Cruz Biotechnology, TRIzol reagent, TaqMan primers and TaqMan PCR Master Mix were purchased from Invitrogen (Carlsbad, CA).

Yang N., Xia Z., Shao N., Li B., Xue L., Peng Y., Zhi F, & Yang Y. (2017). Carnosic acid prevents dextran sulfate sodium-induced acute colitis associated with the regulation of the Keap1/Nrf2 pathway. Scientific Reports, 7, 11036.

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Western Blot Analysis of Protein Expression

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Following treatments, INS-1E cells, rat or human islets were lysed for 30 min at 4°C in a cold lysis buffer as described above, then rapidly sonicated and centrifuged at 12,000 g for 20 min. Supernatants were denatured by boiling them in Laemmli's sample buffer, normalized for protein content by a BCA assay, and equal amounts of proteins (25 μg of protein/lane) resolved by 10% SDS-PAGE. After immunoblotting on nitrocellulose membranes, membranes were cut up into 3 or 4 pieces according to the molecular weights profile, in order to observe simultaneously different proteins on the same blot. Each piece was incubated with primary then secondary antibodies, and proteins were visualized by chemiluminescence detection, as previously described [15] (link). Band densities were quantitatively analyzed by Image J (NIH, USA) and normalized to actin, used as our loading control. Anti-cleaved caspase-3, anti-PARP (Poly-ADP-ribose-polymerase), anti-ubiquitin, anti-CHOP (C/EBP-homologous protein), and anti-rabbit IgG antibodies were from Cell-Signaling (Ozyme, St-Quentin-en-Yvelines, France). Anti-proteasome 20S-β5 subunit and anti-mouse IgG antibodies were from Santa-Cruz (Tebu-bio, Le-Perray-en-Yvelines, France). Anti-β-actin was from Sigma-Aldrich (St-Quentin-Fallavier, France).

Broca C., Varin E., Armanet M., Tourrel-Cuzin C., Bosco D., Dalle S, & Wojtusciszyn A. (2014). Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets. PLoS ONE, 9(3), e92066.

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Investigating UCHL1 and Cell Cycle Regulation

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Anti-cyclin B1 from Santa Cruz (sc-245) was used for immunohistochemistry, western blot, immunoprecipitation, immunofluorescence and the proximity ligation assay. Anti-UCHL1 from Sigma-Aldrich (HPA005993) was used for immunohistochemistry, immunofluorescence and proximity ligation assay; Anti-UCHL1 from Cell Signaling Technology (11896) was used for western blot; Anti-UCHL1 from R&D Systems (MAB6007) was used for immunoprecipitation. Additional antibodies used for western blot were anti-cyclin D1 (sc-753, Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-cyclin E (sc-481, Santa Cruz Biotechnology Inc.), anti-p53 (48818, Cell Signaling Technology, Danvers, MA, USA), anti-ubiquitin (3936, Cell Signaling Technology), anti-β-actin (4967, Cell Signaling Technology), anti-β-catenin (9562, Cell Signaling Technology), anti-GSK3α/β (5676, Cell Signaling Technology), anti-p21 (sc-397, Santa Cruz Biotechnology Inc.), and anti-p27 (sc-529, Santa Cruz Biotechnology Inc.).
UCHL1 was transiently silenced by transfection with Silencer select siRNAs (s14616 and s14618; Life Technologies, Carlsbad, CA, USA) duplexed with Lipofectamine RNAiMAX (Life Technologies) at a final concentration of 5 nM. Non-targeting Silencer Select siRNA was the negative control (Life Technologies).

Kwan S.Y., Au-Yeung C.L., Yeung T.L., Rynne-Vidal A., Wong K.K., Risinger J.I., Lin H.K., Schmandt R.E., Yates M.S., Mok S.C, & Lu K.H. (2020). Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression. Cancers, 12(1), 118.

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