Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab19857 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
IP Use at an assay dependent concentration.
CHIPseq Use at an assay dependent concentration. PubMed: 20526341
ICC/IF Use a concentration of 1 - 5 µg/ml.
Flow Cyt 1/100. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-Fr 1/200. (see Abreview)
IHC-P 1/250. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 39 kDa).Can be blocked with Human Oct4 peptide (ab20650).
ChIP Use at an assay dependent concentration. PubMed: 18490265

Target

  • FunctionTranscription 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 specificityExpressed 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 similaritiesBelongs to the POU transcription factor family. Class-5 subfamily.
    Contains 1 homeobox DNA-binding domain.
    Contains 1 POU-specific domain.
  • Developmental stageHighly expressed in undifferentiated embryonic stem cells and expression decreases gradually after embryoid body (EB) formation.
  • DomainThe 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 localizationNucleus. 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

Anti-Oct4 antibody - ChIP Grade images

  • Mouse E6 - E6.5 embryos were sectioned and then fixed in paraformaldehyde. Antigen retrieval was performed using citric acid and the sections permeablized and blocked for 1 hour in serum. Embryos were stained with ab19857 (1:500) in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody (1:000) and developed using ABC and then DAB for color. Staining is within the epiblast as expected.

    See Abreview

  • ab19857 staining Oct4 in F9 cells treated with trans-retinoic acid (ab120728The cells were incubated at 37°C for 2 days in media containing different concentrations of ab120728ab96899) at 1:250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • The image shows a colony of differentiating Human Embryonic Stem Cells double-stained with anti-Oct4 antibody ab19857 (green) and Sox17 antibody (red). The nuclei of Oct-4-positive undifferentiated hESCs stained bright green. Staining was restricted to the nuclei. Oct4-positive nuclei were Sox17-negative, and vice versa. This antibody can be used as a marker of Oct4-positive undifferentiated Human Embryonic Stem Cells.

  • ICC/IF image of ab19857 stained Mouse Embryonic Stem cells. The cells were PFA fixed (4% PFA, 20 min) and incubated with the antibody (ab19857, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue).

    The large nuclei of the Feeder cells can be seen in the image; ab19857 does not localise to these nuclei. ab19857 can be seen localising to the much smaller nuclei of the Mouse Embryonic Stem cells.

  • ab19857 staining OCT4 on genital ridges from E13.5 mouse embryos by immunohistochemistry. The tissue was paraformaldehyde fixed and permeabilized in 0.1% Triton prior to blocking in 1% BSA for 30 minutes at 20°C. The primary antibody was diluted 1:100 and incubated with the sample for 16 hours at 4°C. An Alexa Fluor 568 conjugated goat anti-rabbit antibody, diluted 1:300, was used as the secondary. Nuclear staining shown with DAPI.

    See Abreview

  • ICC/IF image of ab19857 stained mouse embryonic stem cells. The cells were 4% formalin fixed (10 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 (ab19857, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1:00 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).

  • Overlay histogram of Oct4 staining in mouse embryonic stem cells using ab19857 at 1:100 dilution. Purple histogram represents negative control (rabbit IgG); green line represents anti-Oct4 antibody (ab19857).

    See Abreview

  • Oct4 was immunoprecipitated using 0.5mg E14Tg2a whole cell extract, 5µg of Rabbit polyclonal to Oct4 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, E14Tg2a whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19857.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 48kDa: Oct4.

  • All lanes : Anti-Oct4 antibody - ChIP Grade (ab19857) at 1 µg/ml

    Lane 1 : MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (ab27198)
    Lane 2 : MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (ab27196)
    Lane 3 : IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (ab27202)
    Lane 4 : F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (ab27193)
    Lane 5 : MEL-1 (Human embryonic stem cell, male cell line) Whole Cell Lysate (ab27198) with Human Oct4 peptide (ab20650) at 1 µg/ml
    Lane 6 : MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (ab27196) with Human Oct4 peptide (ab20650) at 1 µg/ml
    Lane 7 : IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (ab27202) with Human Oct4 peptide (ab20650) at 1 µg/ml
    Lane 8 : F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (ab27193) with Human Oct4 peptide (ab20650) at 1 µg/ml

    Lysates/proteins at 10 µl per lane.


    Observed band size : 48 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 65 kDa. We are unsure as to the identity of these extra bands.

    MEL-1 and MEL-2 Human Embryonic Stem Cell Lysates (ab27198 and ab27196), IOUD2 Oct4-expressing Mouse Embryonic Stem Cell Lysate (ab272020) and F9 Mouse Embryonic Carcinoma Cell Lysate (ab27193) all contain Oct4 protein. As expected, a band corresponding to Oct4 was detected in all four of the lysates by Westen Blot using anti-Oct4 antibody ab19857. Each of the Oct4 bands was specifically blocked using the immunising peptide of ab19857. The origin of the additional 65 kDa band detected in Lane 3 is unknown and may be a non-specific band that is recognised, in addition to Oct4, by ab19857 in mouse embryonic stem cells.

  • All lanes : Anti-Oct4 antibody - ChIP Grade (ab19857) at 1 µg/ml

    Lane 1 : Human embryonic stem cell lysate
    Lane 2 : Mouse embryonic stem cell lysate
    Lane 3 : Mouse embryonic germ cell lysate
    Lane 4 : Human embryonic stem cell lysate with Human Oct4 peptide (ab20650)
    Lane 5 : Mouse embryonic stem cell lysate with Human Oct4 peptide (ab20650)
    Lane 6 : Mouse embryonic germ cell lysate with Human Oct4 peptide (ab20650)

    Lysates/proteins at 20 µg per lane.


    Predicted band size : 39 kDa
    Observed band size : 43 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 55 kDa (possible glycosylated form,cross reactivity).

    Anti-Oct4 antibody ab19857 only detected a band corresponding to the expected size of Oct4 in human ES cell lysate. In mouse ES and EG cell lysates, ab19857 detected a band of approximately 55 kDa in addition to the expected 39 kDa Oct4 band.

References for Anti-Oct4 antibody - ChIP Grade (ab19857)

This product has been referenced in:

See all 87 Publications for this product

Product Wall

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Human fibromatosis)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Permeabilization Yes - Tween-20
Specification Human fibromatosis
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative Formaldehyde
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Submitted Apr 29 2015

Application Flow Cytometry
Sample Human Cell (iCell® iPSC Diff Day 5)
Permeabilization Yes - 0.25% Triton X-100 in DPBS
Gating Strategy negative control cells
Specification iCell® iPSC Diff Day 5
Preparation Cell harvesting/tissue preparation method: 1 mM EDTA/1 mM EGTA in PBS (calcium- and magnesium-free)
Sample buffer: DPBS
Fixation Acetone
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Submitted Apr 28 2015

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (H9 hESCs)
Permeabilization Yes - 0.25% Triton X-100 in DPBS
Specification H9 hESCs
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
Fixative Paraformaldehyde
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Submitted Apr 28 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (mice pancreas with ELA::Myc tumors)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Permeabilization Yes - Tween-20
Specification mice pancreas with ELA::Myc tumors
Blocking step Serum as blocking agent for 21 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative Formaldehyde
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Submitted Apr 15 2015

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Sample Dog Tissue sections (MDCK pellets in paraffin)
Specification MDCK pellets in paraffin
Permeabilization Yes - Tween-20
Fixative Formaldehyde
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Submitted Mar 04 2015

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Tissue lysate - whole (1-wk-old mouse cerebellum)
Specification 1-wk-old mouse cerebellum
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Oct 03 2014

Application Western blot
Loading amount 35 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Mouse Cell lysate - whole cell (mES)
Specification mES
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
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Submitted Feb 27 2014

Application Western blot
Loading amount 5 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Human Cell lysate - whole cell (undifferentiated human iPS cells)
Specification undifferentiated human iPS cells
Blocking step 5% milk in TBST as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Submitted Jan 22 2014

Application ChIP
Detection step Real-time PCR
Sample Mouse Cell lysate - whole cell (Mouse ES cells)
Specification Mouse ES cells
Negative control Rabbit IgG Negative control genomic regions
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: 1% Formaldehyde
Positive control Known Oct4 binding regions
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Submitted Dec 06 2013

We have added a FITC conjugated version of ab19857 to the catalog as ab171009. Links to both antibodies are given below for your reference:

ab171009 (FITC conjugated)
http://www.abcam.com/oct4-antibody-fitc-ab171009.html

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