Overview

  • Product name
    Anti-Ubiquitin (linkage-specific K48) antibody [EP8589]
    See all Ubiquitin primary antibodies
  • Description
    Rabbit monoclonal [EP8589] to Ubiquitin (linkage-specific K48)
  • Host species
    Rabbit
  • Specificity
    ab140601 only recognizes polyubiquitin chains formed by Lys-48 (K48) residue linkage. This antibody can detect the target in mouse and rat cell lines and induced tissues.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, Flow Cyt, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to residues in Human Polyubiquitin-C.

  • Positive control
    • K48-linked-Ub2-7 This antibody gave a positive result when used in the following methanol fixed cell lines: MCF-7
  • General notes

    The mouse and rat recommendation is based on the WB results. This antibody may not be suitable for IHC with mouse or rat samples.

    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 ab140601 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
ICC/IF 1/500.
IHC-P 1/250.
Flow Cyt 1/20 - 1/1000.

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

 

WB 1/200 - 1/10000. Detects a band of approximately 17-60 kDa (predicted molecular weight: 77 kDa).

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

  • Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution + K48-linked-Ub2-7recombinant protein lysate at 15 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 77 kDa



    Blocking and diluting buffer: 5% NFDM/TBST. This image is produced useing purified ab140601.

  • Purified ab140601 staining Ubiquitin (linkage-specific K48) in MCF7 (Human breast adenocarcinoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a counterstain for primary antibody ab133645 at 1/2000. DAPI was used as a nuclear counterstain and PBS as a negative control.

  • Purified ab140601 staining Ubiquitin (linkage-specific K48) in human endometrium carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

    Negative control 1: PBS in place of primary antibody.

  • All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 200 µg

    Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
    Lane 2 : 293 (Human embryonic kidney epithelial cell) whole cell lysate
    Lane 3 : Mouse heart lysate
    Lane 4 : Rat heart lysate
    Lane 5 : C2C12 (Mouse myoblasts myoblast) whole cell lysate
    Lane 6 : C6 (Rat glial tumor glial cell) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 77 kDa



    Blocking and diluting buffer: 5% NFDM/TBST. This image is produced useing purified ab140601.

  • All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

    Lane 1 : PC-12
    Lane 2 : C6
    Lane 3 : L6
    Lane 4 : C2C12
    Lane 5 : Neuro-2a
    Lane 6 : NIH3T3
    Lane 7 : SP2/0
    Lane 8 : Raw264.7
    Lane 9 : B16-F0

    Lysates/proteins at 20 µg per lane.

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

    Predicted band size: 77 kDa



    Observed band is above 60kDa

  • All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/200 dilution

    Lane 1 : HEK293
    Lane 2 : Jurkat

    Lysates/proteins at 10 µg per lane.

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

    Predicted band size: 77 kDa



    Observed band is above 60kDa 

  • All lanes : Anti-Ubiquitin (linkage-specific K48) antibody [EP8589] (ab140601) at 1/1000 dilution

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

    Lysates/proteins at 0.01 µg per lane.

    Secondary
    All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

    Predicted band size: 77 kDa
    Observed band size: 17-60 kDa
    why is the actual band size different from the predicted?

  • ICC/IF image of ab140601 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab140601 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Overlay histogram showing HeLa cells stained with ab140601 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab140601, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

References

This product has been referenced in:
  • Sato K  et al. Partial proteasomal degradation of Lola triggers the male-to-female switch of a dimorphic courtship circuit. Nat Commun 10:166 (2019). Read more (PubMed: 30635583) »
  • Zhang L  et al. Cysteine-rich intestinal protein 1 suppresses apoptosis and chemosensitivity to 5-fluorouracil in colorectal cancer through ubiquitin-mediated Fas degradation. J Exp Clin Cancer Res 38:120 (2019). Read more (PubMed: 30850009) »
See all 34 Publications for this product

Customer reviews and Q&As

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1-7 of 7 Abreviews

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - 0.5% Triton X-100
Specification
HeLa
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Nov 02 2018

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
fish gelatine buffer with 0.1% casein and 5% goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 20°C
Sample
Mouse Cell (immortalised bone marrow-derived macrophages)
Specification
immortalised bone marrow-derived macrophages
Permeabilization
Yes - 0.1% Triton X-100
Fixative
Paraformaldehyde

Dr. James Harris

Verified customer

Submitted Sep 08 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (4-12)
Sample
Mouse Cell lysate - whole cell (MEFs)
Specification
MEFs
Treatment
-/+ 4 h MG132 (25 μM)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Nov 19 2013

Application
Western blot
Loading amount
0.03 µg
Gel Running Conditions
Reduced Denaturing (4-12)
Sample
Human Recombinant protein (Recombinant protein)
Specification
Recombinant protein
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Nov 19 2013

Application
Western blot
Loading amount
0.01 µg
Gel Running Conditions
Reduced Denaturing
Sample
Human Recombinant protein (recombinant protein)
Specification
recombinant protein
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 Nov 12 2013

Application
Western blot
Sample
Mouse Tissue lysate - whole (lung)
Loading amount
50000 cells
Specification
lung
Treatment
ip injection with PBS or 1 mg/kg bortezomib overnight
Gel Running Conditions
Reduced Denaturing (4-20% Bis-Tris)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Feb 01 2013

Application
Western blot
Sample
Human Cell lysate - whole cell (lung cancer cells)
Loading amount
10 µg
Specification
lung cancer cells
Treatment
with DMSO or 100 nM bortezomib for 24 hours
Gel Running Conditions
Reduced Denaturing (4-20% Bis-Tris)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jan 24 2013

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