Overview

  • Product name

    Anti-Oct4 antibody [EPR17929] - Low endotoxin, Azide free
    See all Oct4 primary antibodies
  • Description

    Rabbit monoclonal [EPR17929] to Oct4 - Low endotoxin, Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, ChIP, IP, IHC-Fr, Flow Cytmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human Oct4 aa 1-100. The exact sequence is proprietary.
    Database link: Q01860

  • Positive control

    • WB: NCCIT, F9, and NTERA-2 cl.D1 whole cell lysates. IHC-P: Human seminoma and dysgerminoma of ovary tissues. ICC/IF: NCCIT cells. IP: NCCIT whole cell extract. ChIP: Chromatin prepared from F9 cells.
  • General notes

    ab215386 is a carrier-free antibody designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our Low endotoxin, azide-free formats have low endotoxin level (≤ 1 EU/ml, determined by the LAL assay) and are free from azide, to achieve consistent experimental results in functional assays.

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab215386 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
WB Use at an assay dependent concentration. Detects a band of approximately 45 kDa (predicted molecular weight: 39 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

Target

  • Function

    Transcription factor that binds to the octamer motif (5'-ATTTGCAT-3'). Forms a trimeric complex with SOX2 on DNA and controls the expression of a number of genes involved in embryonic development such as YES1, FGF4, UTF1 and ZFP206. Critical for early embryogenesis and for embryonic stem cell pluripotency.
  • Tissue specificity

    Expressed in developing brain. Highest levels found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. Low levels of expression in adult tissues.
  • Sequence similarities

    Belongs to the POU transcription factor family. Class-5 subfamily.
    Contains 1 homeobox DNA-binding domain.
    Contains 1 POU-specific domain.
  • Developmental stage

    Highly expressed in undifferentiated embryonic stem cells and expression decreases gradually after embryoid body (EB) formation.
  • Domain

    The POU-specific domain mediates interaction with PKM2.
  • Post-translational
    modifications

    Sumoylation enhances the protein stability, DNA binding and transactivation activity. Sumoylation is required for enhanced YES1 expression.
    Ubiquitinated; undergoes 'Lys-63'-linked polyubiquitination by WWP2 leading to proteasomal degradation.
  • Cellular localization

    Nucleus. Expressed in a diffuse and slightly punctuate pattern.
  • Information by UniProt
  • Database links

  • Alternative names

    • Octamer binding transcription factor 4 antibody
    • MGC22487 antibody
    • Oct 3 antibody
    • Oct 4 antibody
    • Oct-3 antibody
    • Oct-4 antibody
    • OCT3 antibody
    • Oct4 antibody
    • Octamer binding protein 3 antibody
    • Octamer binding protein 4 antibody
    • Octamer binding transcription factor 3 antibody
    • Octamer-binding protein 3 antibody
    • Octamer-binding protein 4 antibody
    • Octamer-binding transcription factor 3 antibody
    • OTF 3 antibody
    • OTF 4 antibody
    • OTF-3 antibody
    • OTF3 antibody
    • OTF4 antibody
    • PO5F1_HUMAN antibody
    • POU class 5 homeobox 1 antibody
    • POU domain class 5 transcription factor 1 antibody
    • POU domain transcription factor OCT4 antibody
    • POU domain, class 5, transcription factor 1 antibody
    • POU-type homeodomain-containing DNA-binding protein antibody
    • POU5F1 antibody
    see all

Images

  • ab181557 staining Oct4 in Human embryonic stem cells by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with formaldehyde , permeabilized with 0.1% Triton in PBS for 1 hour and blocked with 10% Serum for 1 hour at 25°C. Samples were incubated with primary antibody (1/200 in PBS with 0.1% Tween20) for 16 hours at 4°C. A monoclonal Goat Anti-rabbit Alexa Fluor® 594  was used as the secondary antibody at 1/200 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

  • ab181557 staining OCT-4 in the human cell line NCCIT (human pluripotent embryonal carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/70. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody (Red).

    Isotype control: Rabbit IgG monoclonal [EPR25A] ab172730 (Black).

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

  • ab181557 staining Oct4 in mouse testis (postnatal day 6) tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked with 3% BSA for 30 minutes at 20°C. Samples were incubated with primary antibody (1/1000) for 1 hour at 20°C. Ab150081 (1/500) was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

  • Immunohistochemical analysis of paraffin-embedded Human seminoma tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on cancer cells of Human seminoma is observed. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Human dysgerminoma of ovary tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear and weak cytoplasmic staining on cancer cells of Human dysgerminoma of ovary is observed. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on Human breast cancer. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on adult Human testis. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on adult mouse kidney. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Oct4 with ab181557 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Negative staining on adult rat testis. Counter stained with Hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCCIT (Human pluripotent embryonic carcinoma) cells (positive cell line) or NIH/3T3 (Mouse embyro fibroblast) cells (negative cell line) labeling Oct4 with ab181557 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing nuclear and weakly cytoplasmic staining on NCCIT cell line. Negative expression in NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab181557 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 1/400 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

  • Oct4 was immunoprecipitated from 1mg of NCCIT (Human pluripotent embryonic carcinoma) whole cell extract with ab181557 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab181557 at 1/10000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

    Lane 1: NCCIT whole cell extract 10 µg (Input). Lane 2: ab181557 IP in NCCIT whole cell extract. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab181557 in NCCIT whole cell extract.
    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 3 minutes

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

  • Chromatin was prepared from F9 (Mouse embyro testicular cancer cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab181557 (blue), and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

    “pro” stands for promoter region, while “NC2” stands for negative control which is negative loci at the promoter region.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab181557).

References

ab215386 has not yet been referenced specifically in any publications.

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