Anti-Ubiquitin antibody [Ubi-1] (ab7254)

Mouse monoclonal Ubiquitin antibody [Ubi-1]. Validated in WB, ELISA, IHC, ICC/IF and tested in Mouse, Rat, Chicken, Cow, Human, Arabidopsis thaliana, Caenorhabditis elegans and more.

Overview

  • Product name
    Anti-Ubiquitin antibody [Ubi-1]
    See all Ubiquitin primary antibodies
  • Description
    Mouse monoclonal [Ubi-1] to Ubiquitin
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC-P, WB, ELISA, ICC/IF, IHC-Frmore details
    Unsuitable for: IP
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Drosophila C virus
  • Immunogen

    Full length protein corresponding to Ubiquitin conjugated to Keyhole Limpet Haemocyanin (KLH). Mouse monoclonal [Ubi-1] to Ubiquitin was raised against purified ubiquitin conjugated with glutaraldehyde to keyhole limpet hemocyanin.

  • Positive control
    • H9C2 cells IHC-P:FFPE mouse brain alzheimer
  • General notes

    This product was changed from ascites to tissue culture supernatant on 17th May 2016. The following lots are from ascites and are still in stock as of 17th May 2016: GR159581-2, GR159581-12, GR159581-13. Lot numbers higher than GR159581-13 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

Properties

Applications

Our Abpromise guarantee covers the use of ab7254 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB 1/100 - 1/5000.
ELISA Use at an assay dependent concentration.
ICC/IF 1/500.
IHC-Fr 1/1000.
  • Application notes
    Is unsuitable for IP.
  • Target

    • Relevance
      Function: Ubiquitin exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in lysosomal degradation; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling. Similarity: Belongs to the ubiquitin family. Contains 3 ubiquitin-like domains.
    • Cellular localization
      Cell Membrane, Cytoplasmic and Nuclear
    • Database links
    • Alternative names
      • Epididymis secretory protein Li 50 antibody
      • FLJ25987 antibody
      • HEL S 50 antibody
      • MGC8385 antibody
      • Polyubiquitin B antibody
      • RPS 27A antibody
      • RPS27A antibody
      • UBA 52 antibody
      • UBA 80 antibody
      • UBA52 antibody
      • UBA80 antibody
      • UBB antibody
      • UBB_HUMAN antibody
      • UBC antibody
      • UBCEP 1 antibody
      • UBCEP 2 antibody
      • UBCEP1 antibody
      • UBCEP2 antibody
      • Ubiquitin antibody
      • Ubiquitin B antibody
      see all

    Images

    • All lanes : Anti-Ubiquitin antibody [Ubi-1] (ab7254) at 1/100 dilution

      Lane 1 : HeLa cell lysate
      Lane 2 : HeLa cell lysate with MG132
      Lane 3 : Mouse brain tissue lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Anti mouse IgG at 1/10000 dilution
    • IHC image of ab7254 staining in mouse alzheimer brain formalin fixed paraffin embedded tissue section, using MOM detection kit, ab127055. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab7254, 5µg/ml, for 15 mins at room temperature.  DAB was used as the chromogen (ab103723). The section was counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    • Immunohistochemical staining of ubiquitin in a 7 months-old MPS IIIA mouse brain (PFA fixed) using ab7254.

      Sections, 40 µm thick, were cut sagittally on a vibratome. Slices were permeabilized in 1% triton X-100/PBS for 1 hour at room temperature. The samples were placed on vectabond-coated glass slides and dried overnight. They were then rehydrated in PBS and washed in ice-cold methanol for 10 min.

      The slices were washed in 0.3% H2O2 in methanol at room temperature and incubated sequentially with 0.5% normal donkey serum, ab7254 and biotin-labeled F(ab')2 donkey secondary antibody in Tris-buffered saline (pH 7.5) containing 2% BSA and 0.02% Tween 20. The signal was detected with an ABC kit, visualized with diaminobenzidine and counterstained with hematoxylin.

    • Human normal placenta (ab29745). Staining is localised in the cytoplasm and in the nuclei. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus, at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

    • HeLa cells were co-transfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6 hours (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with ab7254 at a 1/2000 dilution. The secondary used was an Alexa-Fluor 680 conjugated goat anti-mouse polyclonal used at a 1/10000 dilution. The protein is known to be degraded through proteasome.

      See Abreview

    References

    This product has been referenced in:
    • Martínez-Puente DH  et al. Targeting HPV-16 antigens to the endoplasmic reticulum induces an endoplasmic reticulum stress response. Cell Stress Chaperones 24:149-158 (2019). Read more (PubMed: 30604352) »
    • Ren CC  et al. Effects of shRNA-mediated silencing of PSMA7 on cell proliferation and vascular endothelial growth factor expression via the ubiquitin-proteasome pathway in cervical cancer. J Cell Physiol 234:5851-5862 (2019). Read more (PubMed: 29247526) »
    See all 43 Publications for this product

    Customer reviews and Q&As

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    1-7 of 7 Abreviews

    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Cardiomyocytes)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    25 µg
    Treatment
    Starvation
    Specification
    Cardiomyocytes
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

    Abcam user community

    Verified customer

    Submitted Nov 11 2016

    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: RT°C
    Sample
    Human Cell (Human primary fibroblasts)
    Specification
    Human primary fibroblasts
    Permeabilization
    Yes - 0.2% Triton-X100
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Mar 26 2014

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Drosophila C Virus Cell (fly heart tube)
    Specification
    fly heart tube
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 1% triton-x100
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

    Abcam user community

    Verified customer

    Submitted Feb 11 2013

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cells)
    Loading amount
    100 µg
    Specification
    HeLa cells
    Treatment
    10 µM MG-132 for 6hrs
    Gel Running Conditions
    Reduced Denaturing (10% Tris-Gly gel)
    Blocking step
    LI-COR® Odyssey® Blocking Buffer as blocking agent for 45 minute(s) · Concentration: 50% · Temperature: RT°C

    Abcam user community

    Verified customer

    Submitted Dec 13 2011

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa Cells)
    Loading amount
    20 µg
    Specification
    HeLa Cells
    Gel Running Conditions
    Non-reduced Denaturing (10% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

    Abcam user community

    Verified customer

    Submitted Mar 23 2011

    Application
    Western blot
    Sample
    Human Recombinant protein (N-terminal 6His-tagged recombinant Ubiquitin)
    Loading amount
    1 µg
    Specification
    N-terminal 6His-tagged recombinant Ubiquitin
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

    Mrs. Luiza GHILA

    Verified customer

    Submitted Nov 13 2006

    This product is known to not work in this application or species.

    Additional data

    Notes
    We used this antibody in Western blotting and immunoprecipitation.
    In the Western blotting: we were able to see higher amount of intracellular ubiquitin in whole cell extracts upon transfection of ubiquitin in 293 cells. This appeared as a smear, rather than distinct bands.
    In immunoprecipitation: we immunoprecipitated proteins that we expected to be ubiquitylated (IkB, p53)with anti-ubiquitin antibody, however we were unable to detect tham by western with appropriate antibodies. This antibody is probably not suitable for IP.,WB: 1000x
    IP: 10ul/1ml of cell extract,WB: 1000x
    IP: 10ul/1ml of cell extract,WB: 1000x
    IP: 10ul/1ml of cell extract,WB: 1000x
    IP: 10ul/1ml of cell extract
    Dilution notes
    WB: 1000x
    IP: 10ul/1ml of cell extract

    jan bohuslav

    Verified customer

    Submitted Apr 13 2006

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