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Anti ubiquitin

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Anti-ubiquitin is a laboratory reagent used for the detection and quantification of ubiquitin, a small regulatory protein found in all eukaryotic cells. It is commonly used in Western blotting, immunoprecipitation, and other protein analysis techniques.

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Lab products found in correlation

Anti flag, by Merck Group (7 mentions) Anti ha, by Cell Signaling Technology (3 mentions) Anti ha, by Merck Group (3 mentions) Anti cytochrome c, by BD (3 mentions) Anti actin, by Merck Group (2 mentions) Anti myc, by Merck Group (2 mentions) Anti fis1, by Abcam (2 mentions) Anti lc3b, by Abcam (2 mentions) Anti mfn2, by Cell Signaling Technology (2 mentions) Anti tom20, by Proteintech (2 mentions) Anti drp1ser616, by Cell Signaling Technology (2 mentions) Anti tim23, by Proteintech (2 mentions) Anti bcl2 l 13, by Proteintech (2 mentions) Anti bcl2 l 13, by Abcam (2 mentions) Anti drp1ser637, by Cell Signaling Technology (2 mentions) Anti atp synthase subunit beta, by Thermo Fisher Scientific (2 mentions) Anti drp1, by Thermo Fisher Scientific (2 mentions) Anti opa1, by BD (2 mentions) Anti mfn1, by Abnova (2 mentions) Anti mcherry, by Takara Bio (2 mentions)

17 protocols using anti ubiquitin

1

In Vitro Deubiquitination Assay of Ataxin-3

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The in vitro deubiquitination assay was performed as previously described.34 (link)
1.2 μg HIS-ataxin-3 proteins were incubated with 0.5 μg K63-linked hexa-ubiquitin chains (Boston Biochem) in buffer containing 50 mM HEPES pH 8, 0.5 mM EDTA, and 1 mM DTT. Incubations were performed for 16 hr at 37°C. Reactions were stopped with 4× Laemmli sample buffer containing β-mercaptoethanol and by boiling the sample at 100°C for 5 min. Samples were run on SDS-PAGE using 10% TGX gels (Bio-Rad). After western blotting, membranes were probed with anti-ataxin-3 1H9 (1:5,000) and anti-ubiquitin 1:1,000 (UG9511) (Enzo Life Sciences).
Toonen L.J., Rigo F., van Attikum H, & van Roon-Mom W.M. (2017). Antisense Oligonucleotide-Mediated Removal of the Polyglutamine Repeat in Spinocerebellar Ataxia Type 3 Mice. Molecular Therapy. Nucleic Acids, 8, 232-242.
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2

Affinity Purification Reagents and Antibodies

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NAC, Vit C, αLA, ATP, CBB-R250, EDTA, FeSO4, glycerol, H2O2, imidazole, NaCl, NaH2PO4, NEM, Nonidet P40, PMSF, puromycin solution, SDS, Tris-HCl, Triton X-100, EZviewTM Red affinity gels (including streptavidin-HRP, anti-myc, and anti-FLAG® M2), anti-actin (1:2000), and anti-FLAG M2 (1 μg/mL) antibodies were from Sigma-Aldrich (St. Luis, MO); USP2, anti-RPN7 (1:1000), and anti-ubiquitin (1:1000) antibodies from Enzo Life Sciences (Farmingdale, NY); anti-RPN6 antibody (1:1000) from Novus Biologicals (Littleton, CO); AcTEV protease, DTT, IPTG, MgCl2, lipofectAMINE®, PLUSTM reagent, and anti-V5 (1:5000) antibodies from Life Technologies (Grand Island, NY); strepavidin-HRP conjugate (1:3000) from GE Healthcare (Piscataway, NJ); Bortezomib from LC Laboratories (Woburn, MA); anti-PCID2 (1:500) and anti-DSS1FL70 (1:500) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); complete EDTA-free protease inhibitor cocktail from Roche (Indianapolis, IN); anti-eIF3C antibody (1:15,000) from Bethyl Laboratories (Montgomery, TX); anti-His (1:500) antibody from EMD Biosciences (San Diego, CA); anti-RPN3 and anti-DSS1s3259-2 (1 μg/mL) antibodies from Proteintech (Chicago, IL); EcoRV, SgfI, and MluI restriction enzymes are from New England Biolabs (Ipswich, MA).
Zhang Y., Chang F.M., Huang J., Junco J.J., Maffi S.K., Pridgen H.I., Catano G., Dang H., Ding X., Yang F., Kim D.J., Slaga T.J., He R, & Wei S.J. (2014). DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells. Protein & Cell, 5(2), 124-140.
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3

Antibody Characterization for Cell Biology

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Anti-Tim23 (1/1,000), anti-Tom20 (for western blots, 1/1,000, for immunofluorescence, 1/200), anti-Bcl2-L-13 (1/1,000) (Proteintech), anti-cytochrome c (1/1,000) (BD biosciences), anti-Bcl2-L-13 (1/500), anti-GAPDH (1/1,000), anti-Fis1 (1/1,000), anti-α-tubulin (1/1,000) (Abcam), anti-LC3B (for western blots, 1/1,000, for immunofluorescence, 1/100), anti-HA (for western blots, 1/1,000, for immunofluorescence, 1/200), anti-Drp1Ser637 (1/500), anti-Drp1Ser616 (1/1,000), anti-Mfn2 (1/1,000), anti-cleaved caspase 3 (1/1,000) (Cell Signaling Technology), anti-ATP synthase subunit beta (for western blots, 1/1,000, for immunofluorescence, 1/200) (Life Technologies), anti-Drp1 (for western blots, 1/1,000, for immunofluorescence, 1/100), anti-Opa1 (1/1,000) (BD Transduction Laboratories), anti-FLAG (for immunofluorescence, 1/200) (Sigma), anti-Mfn1 (1/1,000) (Abnova), anti-ubiquitin (for immunofluorescence, 1/200) (Enzo Life Science), anti-Phospho-Serine/Threonine (Upstate), anti-mCherry (1/500) (Clontech) and anti-RFP23 (link) (1/1,000) antibodies were used in this study.
Murakawa T., Yamaguchi O., Hashimoto A., Hikoso S., Takeda T., Oka T., Yasui H., Ueda H., Akazawa Y., Nakayama H., Taneike M., Misaka T., Omiya S., Shah A.M., Yamamoto A., Nishida K., Ohsumi Y., Okamoto K., Sakata Y, & Otsu K. (2015). Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation. Nature Communications, 6, 7527.
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4

Mitochondrial Dynamics and Apoptosis Regulation

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Anti-Tim23 (1/1000), anti-Tom20 (for western blots, 1/1000, for immunofluorescence, 1/200), anti-Bcl2-L-13 (1/1000) (Proteintech), anti-cytochrome c (1/1000) (BD biosciences), anti-Bcl2-L-13 (1/500), anti-GAPDH (1/1000), anti-Fis1 (1/1000), anti-α-tubulin (1/1000) (Abcam), anti-LC3B (for western blots, 1/1000, for immunofluorescence, 1/100), anti-HA (for western blots, 1/1000, for immunofluorescence, 1/200), anti-Drp1Ser637 (1/500), anti-Drp1Ser616 (1/1000), anti-Mfn2 (1/1000), anti-cleaved caspase 3 (1/1000) (Cell Signalling Technology), anti-ATP synthase subunit beta (for western blots, 1/1000, for immunofluorescence, 1/200) (Life Technologies), anti-Drp1 (for western blots, 1/1000, for immunofluorescence, 1/100), anti-Opa1 (1/1000) (BD Transduction Laboratories), anti-FLAG (for immunofluorescence, 1/200) (Sigma), anti-Mfn1 (1/1000) (Abnova), anti-ubiquitin (for immunofluorescence, 1/200) (Enzo Life Science), anti-Phospho-Serine/Threonine (Upstate), anti-mCherry (1/500) (Clontech) and anti-RFP23 (link) (1/1000) antibodies were used in this study.
Murakawa T., Yamaguchi O., Hashimoto A., Hikoso S., Takeda T., Oka T., Yasui H., Ueda H., Akazawa Y., Nakayama H., Taneike M., Misaka T., Omiya S., Shah A.M., Yamamoto A., Nishida K., Ohsumi Y., Okamoto K., Sakata Y, & Otsu K. (2015). Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation. Nature Communications, 6, 7527.
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5

Ubiquitin Immunoprecipitation and Western Blot

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Cells were rinsed twice in ice-cold PBS and lysed in cold lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, and 0.2 mM sodium orthovanadate) containing a protease inhibitor cocktail. Samples were precleared with protein A-agarose. Precleared lysates were incubated with 2 μg of anti-ubiquitin (Enzo Life Science, 5120 Butler Pike Plymouth Meeting, PA) or anti-IgG (Cell signaling) primary antibodies overnight at 4°C. Protein A-agarose was added to the lysate, and the mixture was incubated for 2 to 3 hours with agitation at 4°C and then pelleted by centrifugation at 3000 rpm for 5 minutes. The supernatant was removed, and the pellet was washed four times (twice with lysis buffer, once with TBS, and once with 0.05 M Tris-HCl), boiled for 5 minutes, separated by SDS-PAGE, and transferred onto a nitrocellulose membrane. Membranes were probed with an infrared-conjugated secondary antibody, and the signals were visualized and quantitated using LI-COR Odyssey v3.0 software.
Wang J., Lu R., Fu X., Dan Z., Zhang Y.G., Chang X., Liu Q., Xia Y., Liu X, & Sun J. (2018). Novel Regulatory Roles of Wnt1 in Infection-Associated Colorectal Cancer. Neoplasia (New York, N.Y.), 20(5), 499-509.
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6

Ubiquitination of Trichoplein by Cul3-KCTD17

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A measure of 12 μg of His-Ubiquitin (LifeSensors Inc), 1 μg of MBP-trichoplein, 0.1 μg of Cul3-3xFlag immunocomplex, 1 μg of GST-KCTD17, 0.4 μg of His-E1 (Ube1; ENZO Life Sciences) and 0.5 μg His-E2 (Ubiquitin-conjugating enzyme sampler pack; Enzo Life Sciences) were incubated in 20 μl of reaction mixture (50 mM Tris–HCl (pH7.5), 5 mM MgCl2, 2 mM NaF, 2 mM ATP, 0.6 mM dithiothreitol) at 37 °C for 1 h. The reaction was subjected to immunoprecipitation using anti-trichoplein or anti-MBP before immunoblotting with anti-Ubiquitin.
Kasahara K., Kawakami Y., Kiyono T., Yonemura S., Kawamura Y., Era S., Matsuzaki F., Goshima N, & Inagaki M. (2014). Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nature Communications, 5, 5081.
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7

Quantitative Analysis of Rpb1 Ubiquitylation

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To determine total cellular levels and the level of polyubiquitylation of Rpb1 quantitative western blot analysis was performed. Whole cell extracts or purified RNAPII were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting and probed with anti-Rpb1 (8WG16, Covance), anti-Pgk1 (Invitrogen), anti-ubiquitin (Enzo Life Sciences), linkage-specific anti-ubiquitin (Apu2.07 and Apu3.A8, Genentech) or anti-20S (‘core’ subunits, Enzo Life Sciences) antibodies, respectively. Western blot signals were in the linear range of the antibody reaction, acquired using a Fujifilm Mini-LAS300 System (Fujifilm Life Sciences) and quantified with MultiGauge ScienceLab2005Ver3 (Fujifilm Life Sciences). Levels of Rpb1 were calculated relative to the loading control Pgk1 and ubiquitin signals relative to purified Rpb1 from at least three biologically independent experiments.
Karakasili E., Burkert-Kautzsch C., Kieser A, & Sträßer K. (2014). Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1. Nucleic Acids Research, 42(16), 10503-10515.
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8

In Vitro Ubiquitination of STARD9-MD, EMI1, and GFP

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A 10-μg amount of recombinant GST-tagged STARD9-MD, EMI1, or GFP was bound to magnetic glutathione beads (Thermo Scientific) and incubated with mitotic HeLa cell extracts along with an ATP regeneration system (Enzo Life Sciences) for 3 h at 30°C. After 3 h, a master mix containing all the ubiquitination components (ubiquitin [Enzo Life Sciences], ROC1, E1, E2, Skp1, with or without β-TrCP, with or without CUL1, in a buffer containing 20 mM HEPES, 5 mM NaCl, 5 mM MgCl2, DTT, MG132, and protease and phosphatase inhibitor cocktail (Thermo Scientific) and ATP regeneration system was added to the tubes and further incubated for 90 min at 30°C. The beads were then washed four times with a wash buffer containing 20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 15 mM imidazole, 0.5% Triton-X, β-mercaptoethanol, DTT, MG132, and a protease and phosphatase inhibitor cocktail. The beads were then boiled in 2× Laemmli sample buffer (Bio-Rad) and loaded onto a 4–12% TGX gel (Bio-Rad), followed by Western transfer. The blots were subsequently probed with anti-GST (Abcam, Cambridge, MA) and anti-ubiquitin (Enzo Life Sciences) antibodies.
Senese S., Cheung K., Lo Y.C., Gholkar A.A., Xia X., Wohlschlegel J.A, & Torres J.Z. (2015). A unique insertion in STARD9's motor domain regulates its stability. Molecular Biology of the Cell, 26(3), 440-452.
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9

Western Blot Protein Analysis Protocol

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Protein samples were run on 4% to 15% or 7.5% Criterion-TGX Precast Gels (Bio-Rad). The proteins were transferred onto a methanol-activated Immun-Blot PVDF membrane (Bio-Rad), and the membrane was blocked in 5% Carnation powdered skim milk (Nestle) in 1× TBS-T (20 mM Tris, 150 mM NaCl) for 1 h. Primary antibodies were prepared in 3% bovine serum albumin 1× TBS-T with 0.02% sodium azide to the appropriate dilution: anti-Flag and anti-HA (Sigma) 1:2,000, anti-VP35 (6C5 Kerafast) 1:1,000, anti-NP (provided by Dr. Basler, Mount Sinai), anti-ubiquitin (Enzo) 1:1,000, anti-β-actin (Abcam) 1:2,000, anti-His (Sigma) 1:2,000, and anti-β-tubulin (Sigma) 1:1,000. The next day, the blot was washed 3 times every 5 min using 1× TBS-T before incubation with HRP-conjugated goat-anti-rabbit (GE Healthcare) or goat-anti-mouse (GE health care) or Streptavidin (HRP) (Abcam) for 1 h. The blot was washed in 1× TBS-T and developed using Pierce ECL Western Blotting Substrate (Thermo Fisher) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). For blot quantifications, the area under the curve (AUC) was measured for each band of interest using ImageJ.
Rodríguez-Salazar C.A., van Tol S., Mailhot O., Gonzalez-Orozco M., Galdino G.T., Warren A.N., Teruel N., Behera P., Afreen K.S., Zhang L., Juelich T.L., Smith J.K., Zylber M.I., Freiberg A.N., Najmanovich R.J., Giraldo M.I, & Rajsbaum R. (2024). Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replication. PLOS Biology, 22(2), e3002544.
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10

Comprehensive Antibody Panel in Viral Research

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The following antibodies were utilized in this study: TRIM23 monoclonal antibody (M01) (Abnova), rabbit polyclonal anti-STAT1 (BD Biosciences, USA), anti-STAT2 and anti-YFV E (Santa Cruz Biotechnology, USA), antiphospho-STAT1 (Tyr 701) (Cell Signaling Technology, USA), antiphospho-STAT2 (Tyr 689) (Upstate Biotechnology, USA), anti-HA, anti-FLAG, anti-GFP, anti-tubulin, and anti-actin (Sigma-Aldrich, USA), anti-ubiquitin (Enzo Life Sciences, USA), anti-PKR (Abcam), anti-MDA5 (Enzo AT113), anti-DENV-2 E (Hybridoma Facility of Icahn School of Medicine at Mount Sinai, New York, USA). Rabbit antibody against DENV-2 NS5 was generated in our lab and previously reported (Ashour et al., 2009 (link)). Anti-YFV NS5 antibody (YF17D NS5 C7) was kindly provided by Dr. Charles Rice (Chambers et al., 1990b (link)). Universal IFN-I, human IFN-β, human IFN-λ, and human IFN-γ (PBL Interferon Source, USA) were used at 1000 U/ml unless otherwise specified.
Laurent-Rolle M., Morrison J., Rajsbaum R., Macleod J.M., Pisanelli G., Pham A., Ayllon J., Miorin L., Martinez C., tenOever B.R, & García-Sastre A. (2014). The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by Type I interferon. Cell host & microbe, 16(3), 314-327.
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