Overview

  • Product name

    Oct4 Transcription Factor Assay Kit (Colorimetric)
    See all Oct4 kits
  • Detection method

    Colorimetric
  • Sample type

    Nuclear Extracts
  • Assay type

    Semi-quantitative
  • Sensitivity

    500 ng/well
  • Assay time

    3h 30m
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Product overview

    Oct4 Transcription Factor Assay Kit (Colorimetric) (ab207224) is a high throughput assay to quantify Oct4 activation in nuclear extracts. This assay combines a quick ELISA format with a sensitive and specific non-radioactive assay for transcription factor activation.


    A specific double stranded DNA sequence containing the Oct4 consensus binding site (5’ – ATTTGA A ATGCAAAT – 3’) has been immobilized onto a 96-well plate. Active Oct4 present in the nuclear extract specifically binds to the oligonucleotide. Oct4 is detected by a primary antibody that recognizes an epitope of Oct4 accessible only when the protein is activated and bound to its target DNA. An HRP-conjugated secondary antibody provides sensitive colorimetric readout that at OD 450 nm. This product detects human, mouse and rat Oct4.


    Key performance and benefits:



    • Assay time: 3.5 hours (cell extracts preparation not included).

    • Detection limit: < 0.5 µg nuclear extract/well.

    • Detection range: 0.5 – 5 µg nuclear extract/well.

  • Notes

    Oct4 (Octamer Binding Transcription Factor 4) is part of the POU family, whose members contain a bipartite DNA-binding domain consisting of the POU-specific (POUS) and POU homeo-domain (POUHD). Connection of these subdomains by a flexible linker gives POU transcription factors the intrinsic ability to adopt several binding configurations on DNA. POU transcription factors were originally identified as proteins able to activate genes bearing cis-acting elements containing an octameric sequence within their promoter region. They bind to the consensus sequence ATGCAAAT.

    The Oct4 gene has only been found in mammals. In mice, it is located on chromosome 17 and is separated into five exons, which encode a mRNA transcript of 1.5 kb. The human Oct4 is 87% identical to that of the mouse, and is located on chromosome 6. In humans, two transcripts are produced through alternative splicing, while mice produce only one transcript. Oct4 has also been found in other mammalian species including bovine and rat. Oct4 mRNA and protein are present in unfertilized oocytes and expression is abundant and uniform in all cells of the embryo through the morula stage (32-64 cells). After day 7.5, Oct4 is strictly confined to the primordial germ cells (PGCs), precursors of the gametes. Oct4 is also expressed in embryonic stem (ES) and embryonic carcinoma (EC) cells.

    The first morphological indication of differentiation in mammalian embryogenesis is the formation of the trophectoderm (TE) at the early blastocyst stage. While TE cells are restricted to extra-embryonic cell lineages, inner cell mass (ICM) cells remain totipotent. During this phase, Oct4 is downregulated in cells that form the TE, while the cells in the ICM maintain a high level of Oct4. Consequently, a high level of Oct4 expression is thought to keep cells in a totipotent stage, whereas down-regulation is associated with differentiation. Several genes have been found to contain Oct4 binding sites; some positively and some negatively regulated. The α and β subunits of human chorionic gonadotrophin (HGC) are negatively regulated by Oct4 while platelet-derived growth factor (PDGF) α is activated by Oct4.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests 5 x 96 tests
    10X Antibody Binding Buffer 1 x 2.2ml 1 x 11ml
    10X Wash Buffer 1 x 22ml 1 x 110ml
    96-well assay plate 1 unit 5 units
    Anti-rabbit HRP-conjugated IgG (0.25 μg/μL) 1 x 11µl 1 x 55µl
    Binding Buffer 1 x 10ml 1 x 50ml
    Developing Solution 1 x 11ml 1 x 55ml
    Dithiothreitol (DTT) (1 M) 1 x 100µl 1 x 500µl
    Herring sperm DNA (1 μg/μL) 1 x 100µl 1 x 500µl
    Lysis Buffer AM4 1 x 10ml 1 x 50ml
    Mutated oligonucleotide 1 x 100µl 1 x 500µl
    Oct-4 antibody 1 x 11µl 1 x 55µl
    P19 nuclear extract 1 x 40µl 1 x 200µl
    Plate sealer 1 unit 5 units
    Protease Inhibitor Cocktail 1 x 100µl 1 x 500µl
    Stop Solution 1 x 11ml 1 x 55ml
    Wild-type oligonucleotide 1 x 100µl 1 x 500µl
  • Research areas

  • 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
  • Alternative names

    • Octamer binding transcription factor 4
    • MGC22487
    • Oct 3
    • Oct 4
    • Oct-3
    • Oct-4
    • OCT3
    • Oct4
    • Octamer binding protein 3
    • Octamer binding protein 4
    • Octamer binding transcription factor 3
    • Octamer-binding protein 3
    • Octamer-binding protein 4
    • Octamer-binding transcription factor 3
    • OTF 3
    • OTF 4
    • OTF-3
    • OTF3
    • OTF4
    • PO5F1_HUMAN
    • POU class 5 homeobox 1
    • POU domain class 5 transcription factor 1
    • POU domain transcription factor OCT4
    • POU domain, class 5, transcription factor 1
    • POU-type homeodomain-containing DNA-binding protein
    • POU5F1
    see all
  • Database links

Images

  • Different amounts of nuclear extracts from unstimulated HeLa (light grey) and P19 (dark grey) cells were tested for Oct4 activation. These results are provided for demonstration purposes only.

Protocols

References

ab207224 has not yet been referenced specifically in any publications.

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