Anti ubiquitin antibody

Manufactured by Agilent Technologies

The Anti-ubiquitin antibody is a laboratory reagent used to detect and study the presence of ubiquitin, a small regulatory protein found in eukaryotic cells. This antibody can be utilized in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to identify and quantify ubiquitin-conjugated proteins.

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9 protocols using anti ubiquitin antibody

Ubiquitination Assay for SUMO-2 Chains

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Ubiquitination assays were carried out as previously described^{12 (link)}. The following components were mixed together and incubated at room temperature: 0.1 μM E1, 2.5 μM Ubc13, 2.5 μM Ube2V2, 0.55 μM RNF4, 5.5 μM Ub~4xSUMO-2 or 4xSUMO-2, 20 μM Ub, 3 mM ATP, 5 mM MgCl₂, 50 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP, 0.1 % NP40. The reaction was stopped with SDS-PAGE loading buffer and analyzed by SDS-PAGE. Gels were stained with Coomassie Blue. Time points were taken at 0, 2, 5, 10, 20, 40, 60 and 100 min. The zero time point was taken before the addition of ATP. Reactions were also analyzed by western blotting with anti-ubiquitin antibody (Dako).

Branigan E., Plechanovová A., Jaffray E., Naismith J.H, & Hay R.T. (2015). Structural basis for the RING catalyzed synthesis of K63 linked ubiquitin chains. Nature structural & molecular biology, 22(8), 597-602.

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Antibody Panel for NF-κB Signaling

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Antibodies against TRAF1, TRAF2, TRAF3, TRAF6, p50, p52, p105, p100, IκBα, the phosphorylated forms of IKKα (Ser176) β (Ser177), p65 (Ser536), c-jun (Ser63) and IL-6 were purchased from Cell Signaling Technology. Antibodies against p65 and TRAF5 were obtained from Santa Cruz Biotechnology. The antibody against EVER2 was purchased from Interchim. The anti-lamin B antibody was obtained from Thermo Scientific. Recombinant human TNFα was purchased from Miltenyi Biotec. Anti-actin, anti-Flag and anti-hemaglutinin (HA) antibodies, PMA, ionomycin, brefeldin A, Ly294002, SP60025, Bay 11-7082, Gö6976, AG1478, Akt1/2 kinase inhibitor and N-acetylcysteine were obtained from Sigma-Aldrich. Anti-Gluc and anti-Gfp antibodies were obtained from BioLabs. Anti-KL1 antibody was purchased from Immunotech Biotechnologies. Anti-ubiquitin antibody was obtained from Dako.

Vuillier F., Gaud G., Guillemot D., Commere P.H., Pons C, & Favre M. (2014). Loss of the HPV-Infection Resistance EVER2 Protein Impairs NF-κB Signaling Pathways in Keratinocytes. PLoS ONE, 9(2), e89479.

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Ubiquitination Assay for FANCL Proteins

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In a total reaction volume of 1 ml containing assay buffer, 500 mm NaCl, 100 mm Tris, pH 8.0, 250 μm TCEP and 10 μm ZnCl₂, Drosophila FANCL, FANCL L81R, ELF, DRWD-RING, Xenopus FANCL, or FANCL N72R (all 250 nm) was added with an excess of His-ubiquitin (1 μm), His-Ube2T (500 nm) or His-Ube2T-Ub (500 nm). Reactions were left to bind for 1 h on ice. 100 μl of Ni-NTA-agarose (Qiagen) was equilibrated in assay buffer and added to the 1 ml reaction and left on a roller at 4 °C for 1 h. Samples were washed with 10 ml of assay buffer and the agarose resuspended in 100 μl of assay buffer. 50 μl of 2× SDS buffer was added before the samples were boiled. 10 μl of the samples was loaded onto SDS page gel and subjected to Western blotting and probed with appropriate antibodies. Anti-ubiquitin antibody was purchased from DAKO. Anti-Drosophila FANCL and anti-human DRWD antibodies were raised from recombinant proteins by Pettingill Technology Ltd. The anti-His antibody was purchased from GE Healthcare.

Miles J.A., Frost M.G., Carroll E., Rowe M.L., Howard M.J., Sidhu A., Chaugule V.K., Alpi A.F, & Walden H. (2015). The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL. The Journal of Biological Chemistry, 290(34), 20995-21006.

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Antibody Generation and Characterization

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Antibodies against USP11, USP15 and SMAD1 were raised in sheep using GST-tagged proteins as antigens and affinity purified. Anti-SMAD1 antibody for use with Xenopus extracts was from Santa Cruz Biotechnology. Antibody against ALK3 was raised in sheep using His-ALK3(aa200-end) as an antigen and affinity purified. Anti-HA-HRP antibody and anti-α tubulin were from Sigma. Antibodies against phospho-SMAD1/5 (Ser463/465) and 8 (Ser426/428), GAPDH, β-actin and Lamin A/C were from Cell Signaling Technology. Anti-ubiquitin antibody was from Dako. Goat anti-rabbit, mouse and sheep HRP conjugated antibodies were from Pierce. Goat anti-mouse and anti-rabbit IRDye 680LT and 800CW coupled antibodies were from Li-Cor.

Herhaus L., Al-Salihi M.A., Dingwell K.S., Cummins T.D., Wasmus L., Vogt J., Ewan R., Bruce D., Macartney T., Weidlich S., Smith J.C, & Sapkota G.P. (2014). USP15 targets ALK3/BMPR1A for deubiquitylation to enhance bone morphogenetic protein signalling. Open Biology, 4(5), 140065.

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Neuropathological Analysis of Brain Tissues

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At autopsy, brain, spinal cord and other organs were fixed in 10% formalin solution. Samples of the main representative regions of each organ were embedded in paraffin and sectioned in 6 -μm thickness, and stained with haematoxylin and eosin and Klüver-Barrera. Skin biopsy, and following immunostaining and immunofluorescence staining were performed as described previously (Sone et al., 2011 (link)) with anti-ubiquitin antibody (Z0458, Dako) and anti-p62 antibody (sc-28359, Santa Cruz Biotechnology). For assessment of intranuclear inclusion frequency, we prepared a coronal section of cerebral hemisphere, including basal ganglia, stained with anti-p62 antibody using the LSAB Kit (Dako). We selected five random microscopic fields under a ×20 objective lens in each gyrus or basal ganglion, counted the number of neurons, astrocytes and p62-positive intranuclear inclusions and assessed the frequency of intranuclear positive neurons and astrocytes. Samples for electron microscopic study were prepared as described previously (Koike et al., 2007 (link)).

Sone J., Mori K., Inagaki T., Katsumata R., Takagi S., Yokoi S., Araki K., Kato T., Nakamura T., Koike H., Takashima H., Hashiguchi A., Kohno Y., Kurashige T., Kuriyama M., Takiyama Y., Tsuchiya M., Kitagawa N., Kawamoto M., Yoshimura H., Suto Y., Nakayasu H., Uehara N., Sugiyama H., Takahashi M., Kokubun N., Konno T., Katsuno M., Tanaka F., Iwasaki Y., Yoshida M, & Sobue G. (2016). Clinicopathological features of adult-onset neuronal intranuclear inclusion disease. Brain, 139(12), 3170-3186.

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Antibody Characterization and Validation

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Anti-Flag and anti-Myc antibodies were from Sigma. Anti-LRRK2 antibody was from Epitomics (MJFF2) or NeuroMab (N138/6). Anti-WSB1 was from Santa Cruz (SC 393200) or Rabbit anti-WSB1 polyclonal antibodies were developed using a specific epitope to either amino acids 1–15 or amino acids 150–166 of WSB1, and affinity purified (Covance). Anti-alpha-synuclein was from BD Transduction Laboratories and anti-ubiquitin antibody was from Dako Cytomation. Antibodies were used at 1:1,000 dilution for western Blots.

Nucifora FC J.r., Nucifora L.G., Ng C.H., Arbez N., Guo Y., Roby E., Shani V., Engelender S., Wei D., Wang X.F., Li T., Moore D.J., Pletnikova O., Troncoso J.C., Sawa A., Dawson T.M., Smith W., Lim K.L, & Ross C.A. (2016). Ubiqutination via K27 and K29 chains signals aggregation and neuronal protection of LRRK2 by WSB1. Nature Communications, 7, 11792.

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Ubiquitination Assay for SUMO-2 Chains

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LUBAC E3 Ligase Activity Assay

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LUBAC E3 ligase activity assays were performed as described40 (link). Briefly, 1 μg anti-HOIP was coupled to 10 μl packed Protein-G sepharose beads in 50 mM Tris-HCl pH 7.5, 0.2% Triton X-100 for 2 h at 4 °C. The beads were washed 3x in lysis buffer and incubated with 1 mg cell extract for 16 h at 4 °C. The beads were washed 3x in 50 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.2 M NaCl, 0.05% (v/v) 2-mercaptoethanol and once in 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂. The E3 ligase reaction was initiated by the addition of 20 μl of 20 mM Tris-HCl pH 7.5, 2 mM DTT, 0.1 μM UBE1, 0.4 μM UbcH7, 10 μM ubiquitin, 5 mM MgCl₂, 2 mM ATP and incubated for 60 minutes at 30 °C. The reactions were terminated by the addition of 5 μl 4x NuPAGE LDS sample buffer and samples were subjected to SDS-PAGE and immunoblotting with anti-ubiquitin antibody (DAKO #Z0458).

McGuire V.A., Ruiz-Zorrilla Diez T., Emmerich C.H., Strickson S., Ritorto M.S., Sutavani R.V., Weiβ A., Houslay K.F., Knebel A., Meakin P.J., Phair I.R., Ashford M.L., Trost M, & Arthur J.S. (2016). Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation. Scientific Reports, 6, 31159.

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Comprehensive Brain Examination Protocol for bvFTD

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For one p.R406W patient with post-mortem brain examination, the protocol started with an 8–16-week fixation period using 10% buffered formalin. Five-micrometre slices were cut and a macroscopic quantification of localized atrophy took place. Samples from the frontal cortex, the temporal neocortex (superior temporal gyrus), the hippocampus, the striate area, the neostriatum, the basal ganglia, the substantia nigra, the thalamus, the mesencephalon, the pons, the medulla oblongata, the cerebellum and the spinal cord were studied. The latter region is not always studied as part of the standard protocol,^{39 (link),40 (link)} but it was done in this case as the phenotype was bvFTD, which has been associated with concomitant motor neuron disease.
Immunohistochemical staining was performed to determine the nature of possible neuronal inclusions. To that end, anti-ubiquitin antibody (Dako), AT8 (for P-tau; Fujirebio Europe), 4G8 (for amyloid β; Signet), anti-FUS antibody (Proteintech Group Inc.), anti-trans-active response DNA binding protein of 43 kDa antibody (Proteintech Group Inc.) and anti-p62 antibody (BD Diagnostics) were used. The anti-FUS antibody is not always used as part of the standard protocol,^{39 (link),40 (link)} but it is often included in bvFTD cases such as this one.

Gossye H., Van Mossevelde S., Sieben A., Bjerke M., Hendrickx Van de Craen E., van der Zee J., De Deyn P.P., De Bleecker J., Versijpt J., van den Ende J., Deryck O., Bourgeois P., Bier J.C., Goethals M., Vandenberghe R., Engelborghs S, & Van Broeckhoven C. (2022). Patients carrying the mutation p.R406W in MAPT present with non-conforming phenotypic spectrum. Brain, 146(4), 1624-1636.

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