Overview

  • Product nameAnti-Oct4 antibody - ChIP GradeSee all Oct4 primary antibodies ...
  • Description
    Rabbit polyclonal to Oct4 - ChIP Grade
  • Tested applicationsIP, CHIPseq, ICC/IF, Flow Cyt, IHC-Fr, IHC-P, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Sheep, Human, Rhesus monkey
    Predicted to work with: Rat
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 300 to the C-terminus of Human Oct4.

    (Peptide available as ab20650.)

  • Positive control
    • Human ES cell lysate

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 dilution. PubMed: 20526341
ICC/IF Use a concentration of 1 - 5 µg/ml.
Flow Cyt 1/100.
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 dilution. 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.

    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 Oct4-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 78 Publications for this product

Product Wall

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab19857.

To check the status of the order please contact o...

Read More
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 Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Fanconi's Anaemia iPSC)
Specification Fanconi's Anaemia iPSC
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
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Submitted Jan 28 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

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - 0.5% Saponin Buffer
Sample Human Cell (H9 Human Embryonic Stem Cells)
Specification H9 Human Embryonic Stem Cells
Gating Strategy FL1/FSC on isotype control
Preparation Cell harvesting/tissue preparation method: Single cell dissociation buffer
Sample buffer: FACS Buffer (5% FBS)
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Submitted Jul 19 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 37°C
Sample Human Cell (Embryonic Stem Cell)
Specification Embryonic Stem Cell
Permeabilization Yes - BD Perm/Wash Buffer
Fixative Paraformaldehyde
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Submitted May 31 2013

Abreviews
Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - BD Perm/Wash Buffer
Sample Human Cell (Embryonic Stem Cell)
Specification Embryonic Stem Cell
Gating Strategy Gating position dependent on isotype control population
Preparation Cell harvesting/tissue preparation method: Trypsinisation
Sample buffer: BD Perm/Wash Buffer
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Submitted May 22 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

We have b...

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Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: acetic acid
Sample Dog Tissue sections (Gonad at 28 days gestation)
Specification Gonad at 28 days gestation
Permeabilization No
Fixative Paraformaldehyde
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Submitted Mar 29 2013

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