Overview

  • Product name
    Anti-Oct4 antibody - ChIP Grade
    See all Oct4 primary antibodies
  • Description
    Rabbit polyclonal to Oct4 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Previous batches of ab19857 have worked well in IHC, but recent batches of this antibody are showing non-specific staining on day 6 mouse testis in our lab (as of Nov 2016).
  • Tested applications
    Suitable for: IP, CHIPseq, ICC/IF, Flow Cyt, 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.

    Read Abcam's proprietary immunogen policy (Peptide available as ab20650.)

  • Positive control
    • WB: Human and mouse ES cell lysates. ICC/IF: Mouse ES cells.

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.

WB Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 39 kDa).
ChIP Use at an assay dependent concentration. PubMed: 18490265

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

  • ab19857 staining Oct4 in F9 cells treated with trans-retinoic acid (ab120728) The cells were incubated at 37°C for 2 days in media containing different concentrations of ab120728 (trans-retinoic acid) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab19857 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1:250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • 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.

    Predicted band size: 39 kDa
    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 (ab27202) 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.

  • 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.

  • 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-iT 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.

  • 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).

  • 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), 55 kDa (possible 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.

  • 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.

References

This product has been referenced in:
  • Pei Y  et al. Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency. PeerJ 6:e4177 (2018). ICC/IF ; Mouse . Read more (PubMed: 29312817) »
  • Akiyama T  et al. Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing. Sci Rep 8:1189 (2018). Human . Read more (PubMed: 29352121) »
See all 278 Publications for this product

Customer reviews and Q&As

1-10 of 73 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Dog Cell (neural precursors)
Permeabilization
Yes - tritonX 0.05%
Specification
neural precursors
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 15 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (IPSC)
Permeabilization
Yes - 0.3% Triton X-100
Specification
IPSC
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 23°C
Fixative
Paraformaldehyde

Jacob Garza

Verified customer

Submitted Jul 27 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

Abcam user community

Verified customer

Submitted Aug 31 2017

Application
IHC - Wholemount
Sample
Mouse Tissue (mouse P6 testis)
Specification
mouse P6 testis

Jin Yue Liao

Verified customer

Submitted Aug 18 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
CyTOF
Sample
Mouse Cell (lung)
Specification
lung

Irit Milman

Verified customer

Submitted Feb 06 2017

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (induced pluripotent cells)
Permeabilization
Yes - 0.5% TX100
Specification
induced pluripotent cells
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Nov 04 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 May 06 2016

Application
Flow Cytometry
Sample
Human Cell (H9 hESCs)
Permeabilization
Yes - 0.25% Triton X-100 in DPBS
Gating Strategy
Fibroblasts (white)
Specification
H9 hESCs
Preparation
Cell harvesting/tissue preparation method: Accutase
Sample buffer: PBS
Fixation
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 24 2015

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (e12.5 embryo primordial germ cells)
Specification
e12.5 embryo primordial germ cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 04 2015

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

Abcam user community

Verified customer

Submitted Apr 29 2015

1-10 of 73 Abreviews or Q&A

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