Overview

  • Product name
    Anti-A RAF antibody [EPR16208]
    See all A RAF primary antibodies
  • Description
    Rabbit monoclonal [EPR16208] to A RAF
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant fragment within Human A RAF aa 150-300. The exact sequence is proprietary.
    Database link: P10398

  • Positive control
    • WB: HeLa, Raji, HT-29 and A375 whole cell lysates; Human fetal heart, fetal kidney, fetal brain and bladder lysates. IHC-P: Human cervix carcinoma and kidney tissues. ICC/IF: HeLa and HEK293 cells. Flow Cyt: HeLa cells.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    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 ab200653 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 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB 1/1000. Detects a band of approximately 68 kDa (predicted molecular weight: 68 kDa).
ICC/IF 1/250.
Flow Cyt 1/100. ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Involved in the transduction of mitogenic signals from the cell membrane to the nucleus.
  • Tissue specificity
    Predominantly in urogenital tissues.
  • Sequence similarities
    Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. RAF subfamily.
    Contains 1 phorbol-ester/DAG-type zinc finger.
    Contains 1 protein kinase domain.
    Contains 1 RBD (Ras-binding) domain.
  • Information by UniProt
  • Database links
  • Alternative names
    • A raf 1 antibody
    • A Raf proto oncogene serine/threonine protein kinase antibody
    • ARAF 1 antibody
    • Araf antibody
    • ARaf proto oncogene serine/threonine protein kinase antibody
    • ARAF_HUMAN antibody
    • ARAF1 antibody
    • Oncogene Araf1 antibody
    • Oncogene PKS2 antibody
    • PKS 2 antibody
    • PKS antibody
    • PKS2 antibody
    • Proto oncogene Pks antibody
    • Proto-oncogene A-Raf antibody
    • Proto-oncogene A-Raf-1 antibody
    • Proto-oncogene Pks antibody
    • RAFA 1 antibody
    • RAFA1 antibody
    • Ras binding protein DA Raf antibody
    • Serine/threonine-protein kinase A-Raf antibody
    • v raf murine sarcoma 3611 viral oncogene homolog antibody
    • v raf murine sarcoma 3611 viral oncogene homolog 1 antibody
    • v raf oncogene homolog 1 (murine sarcoma 3611 virus) antibody
    see all

Images



  • Predicted band size : 68 kDa

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: A RAF knockout HAP1 cell lysate (20 µg)
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: Raji cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab200653 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab200653 was shown to specifically react with A RAF when A RAF knockout samples were used. Wild-type and ProteinX knockout samples were subjected to SDS-PAGE. ab200653 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and) Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling A RAF with ab200653 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

    Cytoplasm staining on HeLa cell line is observed.

    The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab200653 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

     

  • Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling A RAF with ab200653 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

    Cytoplasm staining on Human cervix carcinoma tissue is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.



  • Predicted band size : 68 kDa

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: A RAF knockout HAP1 cell lysate (20 µg)

    Lanes 1 - 2: Merged signal (red and green). Green - ab200653 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.

    This western blot image is a comparison between ab200653 and a competitor's top cited rabbit polyclonal antibody.

  • Flow cytometric analysis of 2% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling A RAF with ab200653 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293 (Human embryonic kidney) cells labeling A RAF with ab200653 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

    Cytoplasm staining on HEK293 cell line is observed.

    The nuclear counter stain is DAPI (blue).

    Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab200653 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
    -ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

     

  • All lanes : Anti-A RAF antibody [EPR16208] (ab200653) at 1/10000 dilution

    Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate
    Lane 2 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

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

    Predicted band size : 68 kDa
    Observed band size : 68 kDa


    Exposure time : 1 minute

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-A RAF antibody [EPR16208] (ab200653) at 1/1000 dilution

    Lane 1 : HT-29 (Human colorectal adenocarcinoma cells) whole cell lysate
    Lane 2 : A375 (Human malignant melanoma) whole cell lysate
    Lane 3 : Human fetal heart lysate
    Lane 4 : Human fetal kidney lysate
    Lane 5 : Human bladder lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size : 68 kDa
    Observed band size : 68 kDa


    Exposure time : 1 minute

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Anti-A RAF antibody [EPR16208] (ab200653) at 1/1000 dilution + Human fetal brain lysate at 10 µg

    Secondary
    Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

    Predicted band size : 68 kDa
    Observed band size : 68 kDa


    Exposure time : 3 minutes

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling A RAF with ab200653 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

    Cytoplasm staining on Human kidney tissue is observed.

    Counter stained with Hematoxylin.

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

References

This product has been referenced in:
  • Faltermeier CM  et al. Functional screen identifies kinases driving prostate cancer visceral and bone metastasis. Proc Natl Acad Sci U S A 113:E172-81 (2016). Read more (PubMed: 26621741) »

See 1 Publication for this product

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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