Anti-Oct4 antibody - ChIP Grade (ab19857)
- Datasheet
- References (283)
- Protocols
Overview
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Product nameAnti-Oct4 antibody - ChIP Grade
See all Oct4 primary antibodies -
DescriptionRabbit polyclonal to Oct4 - ChIP Grade
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Host speciesRabbit
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SpecificityPrevious 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).
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Tested applicationsSuitable for: IP, CHIPseq, ICC/IF, Flow Cyt, WB, ChIPmore details
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Species reactivityReacts 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.
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Positive control
- WB: Human and mouse ES cell lysates. ICC/IF: Mouse ES cells.
Properties
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
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Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
Constituent: 99% PBS
Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help -
Concentration information loading...
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PurityProtein A purified
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Purification notesAffinity Purified
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ClonalityPolyclonal
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IsotypeIgG
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Research areas
Associated products
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Alternative Products
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Alternative Versions
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Assay kits
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ChIP Related Products
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Compatible Secondaries
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Conjugation kits
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Immunizing Peptide (Blocking)
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Isotype control
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Related Buffer
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Related Products
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 |
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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. |
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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
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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.
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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.
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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.
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DomainThe POU-specific domain mediates interaction with PKM2.
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Post-translational
modificationsSumoylation 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
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Database links
- Entrez Gene: 5460 Human
- Entrez Gene: 18999 Mouse
- Entrez Gene: 294562 Rat
- Entrez Gene: 714760 Rhesus monkey
- Omim: 164177 Human
- SwissProt: Q01860 Human
- SwissProt: P20263 Mouse
- SwissProt: Q5TM49 Rhesus monkey
see all -
Alternative names
- Octamer binding transcription factor 4 antibody
- MGC22487 antibody
- Oct 3 antibody
see all
Images
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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.
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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) withHuman Oct4 peptide (ab20650) at 1 µg/ml
Lane 6 : MEL-2 (Human embryonic stem cell, female cell line) Whole Cell Lysate (ab27196) withHuman Oct4 peptide (ab20650) at 1 µg/ml
Lane 7 : IOUD2 (Mouse embryonic stem cell, selected for Oct4 expression cell line) Whole Cell Lysate (ab27202) withHuman Oct4 peptide (ab20650) at 1 µg/ml
Lane 8 : F9 (Mouse embryonic carcinoma cell line) Whole Cell Lysate (ab27193) withHuman 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.
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Flow Cytometry - Anti-Oct4 antibody - ChIP Grade (ab19857)This image is taken from an abreview by Kate Hawkins
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).
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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.
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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).
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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 withHuman Oct4 peptide (ab20650)
Lane 5 : Mouse embryonic stem cell lysate withHuman Oct4 peptide (ab20650)
Lane 6 : Mouse embryonic germ cell lysate withHuman 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.
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Immunocytochemistry/ Immunofluorescence - Anti-Oct4 antibody - ChIP Grade (ab19857)This image is courtesy of Ludovic Vallier, University of Cambridge
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.
Protocols
References
This product has been referenced in:
- Farahani RM et al. Neural microvascular pericytes contribute to human adult neurogenesis. J Comp Neurol 527:780-796 (2019). Read more (PubMed: 30471080) »
- He B et al. Binding of HMGN proteins to cell specific enhancers stabilizes cell identity. Nat Commun 9:5240 (2018). Read more (PubMed: 30532006) »