Recombinant
RabMAb

Recombinant Anti-Eg5 antibody [EPR23276-52] (ab254298)

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Overview

  • Product name

    Anti-Eg5 antibody [EPR23276-52]
    See all Eg5 primary antibodies

  • Description

    Rabbit monoclonal [EPR23276-52] to Eg5

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, IP, WB, IHC-P, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant fragment within Human Eg5 aa 1-400. The exact sequence is proprietary.
    Database link: P52732

  • Positive control

    • WB: Human testis and tonsil tissue lysate. Mouse testis tissue lysate. Jurkat, NCI-H1975, MCF7 and 4T1 whole cell lysate. IHC-P: Human tonsil and testis tissue. Human breast cancer tissue. ICC/IF: HeLa and NIH/3T3 cells. FC: HeLa and NIH/3T3 cells. IP: HeLa and NIH/3T3 whole cell lysate.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

Our Abpromise guarantee covers the use of ab254298 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 1/50.
IP 1/30.
WB 1/1000. Predicted molecular weight: 119 kDa.
IHC-P 1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Flow Cyt 1/600.

Target

  • Function

    Motor protein required for establishing a bipolar spindle. Blocking of KIF11 prevents centrosome migration and arrest cells in mitosis with monoastral microtubule arrays.

  • Involvement in disease

    Defects in KIF11 are the cause of microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR) [MIM:152950]. An autosomal dominant disorder that involves an overlapping but variable spectrum of central nervous system and ocular developmental anomalies. Microcephaly ranges from mild to severe and is often associated with mild to moderate developmental delay and a characteristic facial phenotype with upslanting palpebral fissures, broad nose with rounded tip, long philtrum with thin upper lip, prominent chin, and prominent ears. Chorioretinopathy is the most common eye abnormality, but retinal folds, microphthalmia, and myopic and hypermetropic astigmatism have also been reported, and some individuals have no overt ocular phenotype. Congenital lymphedema, when present, is typically confined to the dorsa of the feet, and lymphoscintigraphy reveals the absence of radioactive isotope uptake from the webspaces between the toes.

  • Sequence similarities

    Belongs to the kinesin-like protein family. BimC subfamily.
    Contains 1 kinesin-motor domain.

  • Post-translational
    modifications

    Phosphorylated exclusively on serine during S phase, but on both serine and Thr-926 during mitosis, so controlling the association of KIF11 with the spindle apparatus (probably during early prophase). Phosphorylated upon DNA damage, probably by ATM or ATR.
    A subset of this protein primarily localized at the spindle pole is phosphorylated by NEK6 during mitosis; phosphorylation is required for mitotic function.

  • Cellular localization

    Cytoplasm. Cytoplasm > cytoskeleton > spindle pole.
  • Information by UniProt

  • Database links

    • Alternative names

      • EG5 antibody
      • HKSP antibody
      • KIF11 antibody
      • KIF11_HUMAN antibody
      • Kinesin family member 11 antibody
      • Kinesin like protein 1 antibody
      • Kinesin-like protein 1 antibody
      • Kinesin-like protein KIF11 antibody
      • Kinesin-like spindle protein HKSP antibody
      • Kinesin-related motor protein Eg5 antibody
      • KNSL1 antibody
      • MCLMR antibody
      • Thyroid receptor-interacting protein 5 antibody
      • TR-interacting protein 5 antibody
      • TRIP-5 antibody
      • TRIP5 antibody
      see all

    Images

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Immunohistochemical analysis of paraffin-embedded human testis tissue labeling Eg5 with 254298 at 1/1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in spermatocytes of human testis is observed.
      The section was incubated with ab254298 for 30 mins at room temperature.
      The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells labeling Eg5 with ab254298 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic and spindle (arrow) staining in NIH/3T3 cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 2 µg/ml dilution.

    • Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

      Lane 1 : Human testis tissue lysate
      Lane 2 : Human tonsil tissue lysate
      Lane 3 : Human lung tissue lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/100000 dilution

      Predicted band size: 119 kDa



      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

      The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

      Negative control: Human lung (PMID: 27279560).

      The minor bands beneath the target band may correspond to degradation.

      Exposure time: 48 seconds.

    • Flow Cytometry - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Flow Cytometry - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labeling Eg5 with ab254298 at 1/600 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

    • Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Eg5 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug with ab254298 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254298 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: NIH/3T3 whole cell lysate 10 ug.

      Lane 2: ab254298 IP in NIH/3T3 whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254298 in NIH/3T3 whole cell lysate.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 3 minutes

      Fresh lysates were used in this IP.

    • Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunoprecipitation - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Eg5 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate 10 ug with ab254298 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254298 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

      Lane 1: HeLa whole cell lysate 10 ug.

      Lane 2: ab254298 IP in HeLa whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254298 in HeLa whole cell lysate.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 3 minutes.

      Fresh lysates were used in this IP.

    • Flow Cytometry - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Flow Cytometry - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Eg5 with ab254298 at 1/600 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

    • Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunocytochemistry/ Immunofluorescence - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Eg5 with ab254298 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic and spindle (arrow) staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

      Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 2 µg/ml dilution.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling Eg5 with ab254298 at 1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Scattered cytoplasmic staining in human breast cancer cells is observed (PMID:29181100).
      The section was incubated with ab254298 for 30 mins at room temperature.
      The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Eg5 antibody [EPR23276-52] (ab254298)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Eg5 with 254298 at 1/4000 (0.17µg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in human tonsil is observed (PMID:25277178).
      The section was incubated with ab254298 for 30 mins at room temperature.
      The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with hematoxylin.

      Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

    • Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

      Lane 1 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
      Lane 2 : NCI-H1975 (human adenocarcinoma lung epithelial cell)
      Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell)
      Lane 4 : 4T1 (mouse mammary gland carcinoma epithelial cell)

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 119 kDa



      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

      The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

      Fresh lysates were tested in this WB.

      The minor bands beneath the target band in lane 1 and lane 4 may correspond to degradation.

      Exposure time: 15 seconds.

    • Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      Western blot - Anti-Eg5 antibody [EPR23276-52] (ab254298)
      All lanes : Anti-Eg5 antibody [EPR23276-52] (ab254298) at 1/1000 dilution

      Lane 1 : Mouse testis tissue lysate
      Lane 2 : Mouse brain tissue lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Predicted band size: 119 kDa



      Blocking and diluting buffer and concentration: 5% NFDM/TBST.

      Negative control: Mouse brain (PMID: 17974955).

      The minor bands beneath the target band may correspond to degradation.

      Exposure time: 48 seconds.

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References

    Publishing research using ab254298? Please let us know so that we can cite the reference in this datasheet.

    ab254298 has not yet been referenced specifically in any publications.

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