Overview

  • Product name
    Anti-Oct4 antibody [EPR17929] - ChIP Grade
    See all Oct4 primary antibodies
  • Description
    Rabbit monoclonal [EPR17929] to Oct4 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Oct4 is highly expressed in undifferentiated embryonic stem cells and cancer stem cell-like cells (PMID: 26013162, 21826175). ab181557 can’t detect the target band in undifferentiated cancer cell lines with low expression level of Oct4, such as HeLa, HEK-293, MDA-MB-231, HepG2, Huh7, HCT-116 and PANC-1 (PMID: 21975933, 29789579, 25625591, 26059097, 23928699, 27344963, 25837691, 29254202, 28854261, 27996162), even at the dilution of 1:200.
  • Tested applications
    Suitable for: WB, ICC/IF, IP, ChIP, IHC-P, Flow Cyt, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat, Sheep, Horse, Cow, Pig, Common marmoset
  • Immunogen

    Synthetic peptide 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

    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 ab181557 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 1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 39 kDa).
ICC/IF 1/250.
IP 1/50.
ChIP Use 5 µg for 25 µg of chromatin.
IHC-P 1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt Use at an assay dependent concentration.

Purified format.

IHC-Fr 1/100 - 1/1000.

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

  • All lanes : Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/2000 dilution

    Lane 1 : NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysates
    Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 3 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
    Lane 4 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysates
    Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
    Lane 6 : Huh7 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
    Lane 7 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates
    Lane 8 : PANC-1 (Human pancreatic epithelioid carcinoma epithelial cell) whole cell lysates

    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: 39 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    Blocking and diluting buffer: 5% NFDM/TBST

    Oct4 is highly expressed in undifferentiated embryonic stem cells and cancer stem cell-like cells (PMID: 26013162, 21826175). ab181557 can’t detect the target band in undifferentiated cancer cell lines with low expression level of Oct4, such as HeLa, HEK-293, MDA-MB-231, HepG2, Huh7, HCT-116 and PANC-1 (PMID: 21975933, 29789579, 25625591, 26059097, 23928699, 27344963, 25837691, 29254202, 28854261, 27996162), even at the dilution of 1:200.

  • 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.

  • 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.

  • 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.

    See Abreview

  • 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.

  • 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.

    See Abreview

  • 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

  • All lanes : Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557) at 1/1000 dilution

    Lane 1 : NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate
    Lane 2 : F9 (Mouse embyro testicular cancer cell line) whole cell lysate
    Lane 3 : NTERA-2 cl.D1 (Human malignant pluripotent embryonic carcinoma) whole cell lysate
    Lane 4 : Mouse testis lysate
    Lane 5 : Human hippocampus lysate
    Lane 6 : Human cerebellum lysate
    Lane 7 : Human testis lysate
    Lane 8 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

    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: 39 kDa
    Observed band size: 45 kDa why is the actual band size different from the predicted?


    Exposure time: 5 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    Low levels of expression in adult tissues

  • 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).

  • 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.

  • 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.

  • 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.

  • 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.

  • 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.

References

This product has been referenced in:
  • Liu B  et al. PD-1/PD-L1 expression in a series of intracranial germinoma and its association with Foxp3+ and CD8+ infiltrating lymphocytes. PLoS One 13:e0194594 (2018). Read more (PubMed: 29617441) »
  • Xu W  et al. Knockdown of HuR represses osteosarcoma cells migration, invasion and stemness through inhibition of YAP activation and increases susceptibility to chemotherapeutic agents. Biomed Pharmacother 102:587-593 (2018). WB . Read more (PubMed: 29597092) »
See all 8 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Cow Cell (in vitro produced preimplantation embryo day 7)
Permeabilization
Yes - 0.5 % Triton X 100
Specification
in vitro produced preimplantation embryo day 7
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 07 2018

Application
IHC - Wholemount
Sample
Mouse Embryo (Wholemount immunostaining of E6.5 mouse embryo)
Specification
Wholemount immunostaining of E6.5 mouse embryo

Dr. Sophie Morgani

Verified customer

Submitted Feb 09 2018

Application
ChIP
Sample
Human Cell lysate - nuclear (hESC)
Negative control
gene desert
Specification
hESC
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 8 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% PFA
Positive control
upstream and promoter region

Abcam user community

Verified customer

Submitted Aug 25 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Human embryonic stem cells)
Permeabilization
Yes - 0.1% Triton in PBS for 1 hour
Specification
Human embryonic stem cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Joanne Lacey

Verified customer

Submitted Aug 07 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (Human embryonic stem cells)
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE gel)
Loading amount
15 µg
Specification
Human embryonic stem cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

Joanne Lacey

Verified customer

Submitted Aug 07 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (human embryonic stem cells)
Permeabilization
Yes - PBS/Triton X-100
Specification
human embryonic stem cells
Blocking step
Donkey serum/BSA (1%) as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jul 04 2017

Application
Flow Cytometry
Sample
Human Cell (Human embryonic stem cells)
Permeabilization
Yes - 70% methanol
Gating Strategy
All
Specification
Human embryonic stem cells
Fixation
Paraformaldehyde

Dr. Kate Hawkins

Verified customer

Submitted Sep 15 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (human embryonic stem cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Specification
human embryonic stem cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Kate Hawkins

Verified customer

Submitted Sep 12 2016

Application
Immunocytochemistry
Sample
Human Cell (human embryonic stem cells)
Permeabilization
Yes - 0.3% tritonX100
Specification
human embryonic stem cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 25°C
Fixative
Paraformaldehyde

Dr. Kate Hawkins

Verified customer

Submitted Sep 08 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Testis, postnatal day 6)
Permeabilization
Yes - 0.1% Triton X-100 in PBS
Specification
Testis, postnatal day 6
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C
Fixative
Paraformaldehyde

Mr. Bryan Niedenberger

Verified customer

Submitted Mar 07 2016

1-10 of 12 Abreviews or Q&A

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