Overview

  • Product name
    Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034]
    See all Ubiquitin primary antibodies
  • Description
    Rabbit monoclonal [EPR17034] to Ubiquitin (linkage-specific K27)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Ubiquitin aa 1-100 (linkage-specific K27). The exact sequence is proprietary.
    Database link: P0CG48

  • Positive control
    • WB: K27-linked-Ub2 recombinant protein. C6, RAW 264.7, PC-12, NIH/3T3, HeLa, 293T and Jurkat whole cell lysates. Rat and Mouse brain, heart, kidney and spleen lysates. IHC-P: Human transitional cell carcinoma of bladder tissue. Mouse and Rat spleen tissue. Flow Cytometry: Jurkat cells.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab181537 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 1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/5000.

Actual Band: 14kDa and above.

Flow Cyt 1/160.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/500.

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

  • Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma)
    labelling Ubiquitin (linkage-specific K27) with purified ab181537 at 1/1000. Cells were fixed with 4% PFA and permeabilized with 0.1% triton X-100. ab150077 Goat anti rabbit IgG (Alexa Fluor® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

  • All lanes : Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537) at 1/5000 dilution

    Lane 1 : K27-linked-Ub2 recombinant protein
    Lane 2 : K6-linked-Ub2 recombinant protein
    Lane 3 : K11-linked-Ub2 recombinant protein
    Lane 4 : K29-linked-Ub2 recombinant protein
    Lane 5 : K33-linked-Ub2 recombinant protein
    Lane 6 : K48-linked-Ub2 recombinant protein
    Lane 7 : K63-linked-Ub2 recombinant protein
    Lane 8 : Ubiquitin recombinant protein
    Lane 9 : Ubiquitin linear recombinant protein

    Lysates/proteins at 0.02 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution


    Blocking/dilution buffer: 5% BSA/TBST.

    Expected MW is ~17kDa (8.5*2)

    The ~30kDa band may be the dimer of K27-linked-Ub2 recombinant protein.

  • All lanes : Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537) at 1/50000 dilution

    Lane 1 : 293T (Human epithelial cells from embryonic kidney) whole cell lysates
    Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Observed band size: 14 & above kDa
    why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% BSA/TBST.

     

  • Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537) at 1/5000 dilution + HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates at 10 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Observed band size: 14 & above kDa why is the actual band size different from the predicted?



    Blocking/dilution buffer: 5% BSA/TBST.

  • All lanes : Anti-Ubiquitin (linkage-specific K27) antibody [EPR17034] (ab181537) at 1/5000 dilution

    Lane 1 : Mouse brain lysates
    Lane 2 : Mouse heart lysates
    Lane 3 : Mouse kidney lysates
    Lane 4 : Mouse spleen lysates
    Lane 5 : Rat brain lysates
    Lane 6 : Rat heart lysates
    Lane 7 : Rat kidney lysates
    Lane 8 : Rat spleen lysates
    Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
    Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
    Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
    Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

    Observed band size: 14 & above kDa why is the actual band size different from the predicted?



    Blocking/dilution buffer: 5% BSA/TBST.

  • Immunohistochemical analysis of paraffin-embedded Human transitional cell carcinoma of bladder labeling Ubiquitin (linkage-specific K27) with ab181537 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on cancer cells of Human bladder transitional cell carcinoma is observed. Counter stained with Hematoxylin.
    Negative control: Used PBS instead of primary antibody.

  • Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Ubiquitin (linkage-specific K27) with ab181537 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on lymphocytes of Mouse spleen is observed. Counter stained with Hematoxylin.
    Negative control: Used PBS instead of primary antibody.

  • Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling Ubiquitin (linkage-specific K27) with ab181537 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on lymphocytes of Rat spleen is observed. Counter stained with Hematoxylin.
    Negative control: Used PBS instead of primary antibody.

  • Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Ubiquitin (linkage-specific K27) with ab181537 at 1/160 dilution (red). The secondary antibody was Goat anti rabbit IgG (FITC) at 1/150 dilution.  The isotype control is Rabbit monoclonal IgG (black) and cell without incubation with primary antibody and secondary antibody is blue.

References

This product has been referenced in:
  • Yin Q  et al. K27-linked ubiquitination of BRAF by ITCH engages cytokine response to maintain MEK-ERK signaling. Nat Commun 10:1870 (2019). Read more (PubMed: 31015455) »
  • Panda S & Gekara NO The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex. Nat Commun 9:4654 (2018). Read more (PubMed: 30405132) »
See all 3 Publications for this product

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