Overview

  • Product name
    Anti-Otx1 + Otx2 antibody - ChIP Grade
    See all Otx1 + Otx2 primary antibodies
  • Description
    Rabbit polyclonal to Otx1 + Otx2 - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    ab21990 is raised against the C-terminal region of the Human Otx2 protein. This product is batch tested in Y79 cell lysate, recombinant Otx1 and overexpressed Otx2. All batches should detect endogenous Otx2, overexpressed Otx2 and recombinant or overexpressed Otx1.
  • Tested applications
    Suitable for: IHC-Fr, ICC/IF, ChIP, WB, IHC-P, IPmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Cow, Human, Xenopus laevis
    Predicted to work with: Zebrafish
  • Immunogen

    Synthetic peptide corresponding to Human Otx2 aa 250 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Database link: P32243
    (Peptide available as ab24347, ab24348)

  • Positive control
    • This antibody gave a positive signal in the following whole cell lysates: Y79.

      This antibody gave a positive signal in the following cell lines: Human Embryonic Stem cells.

      This antibody gave a positive signal in the following tissues: Formalin Fixed Paraffin Embedded Mouse E6 Embryo Normal; Formalin Fixed Paraffin Embedded Mouse E14.5 Embryo Normal

Properties

Applications

Our Abpromise guarantee covers the use of ab21990 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
IHC-Fr Use a concentration of 0.8 µg/ml.
ICC/IF Use a concentration of 0.8 - 4 µg/ml.
ChIP Use at an assay dependent concentration. PubMed: 18057103
WB Use a concentration of 1 µg/ml. Detects a band of approximately 37 kDa (predicted molecular weight: 31.6 kDa).
IHC-P Use at an assay dependent concentration. PubMed: 20150232
IP Use a concentration of 5 µg/ml.

Target

  • Relevance

    Function: Probably plays a role in the development of the brain and the sense organs. Can bind to the BCD target sequence (BTS): 5'-TCTAATCCC-3'.

    Tissue specificity: Expressed in brain.

    Disease: Defects in OTX2 are the cause of microphthalmia syndromic type 5 (MCOPS5) [MIM:610125]. Microphthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. Up to 80% of cases of microphthalia occur in association with syndromes that include non-ocular abnormalities. MCOPS5 patients manifest unilateral or bilateral microphthalmia/clinical anophthalmia and variable additional features including coloboma, microcornea, cataract, retinal dystrophy, hypoplasia or agenesis of the optic nerve, agenesis of the corpus callosum, developmental delay, joint laxity, hypotonia, and seizures.

    Similarity: Belongs to the paired homeobox family. Bicoid subfamily. Contains 1 homeobox DNA-binding domain.

    Developmental stage: Embryo.

  • Database links
  • Alternative names
    • FLJ38361 antibody
    • Homeobox protein OTX 1 antibody
    • Homeobox protein OTX1 antibody
    • Homeobox protein OTX2 antibody
    • MCOPS 5 antibody
    • MCOPS5 antibody
    • MGC15736 antibody
    • MGC45000 antibody
    • Orthodenticle 1 antibody
    • Orthodenticle 2 antibody
    • Orthodenticle homeobox 1 antibody
    • Orthodenticle homeobox 2 antibody
    • Orthodenticle homolog 1 antibody
    • Orthodenticle homolog 2 (Drosophila) antibody
    • Orthodenticle homolog 2 antibody
    • Orthodenticle1 antibody
    • Orthodenticle2 antibody
    • Otx 1 antibody
    • Otx 2 antibody
    • otx1 antibody
    • OTX1_HUMAN antibody
    • otx2 antibody
    • OTX2_HUMAN antibody
    see all

Images

  • Otx1 +2 was immunoprecipitated using 0.5mg Y79 whole cell extract, 5µg of Rabbit polyclonal to Otx1 +2 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, Y79 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab21990.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 37kDa; Otx1 +2, non specific - as present in control (lane 2); 37kDa: We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest.

  • Immunohistochemistry (Frozen sections) analysis of E8.5 mouse embryo brain section labeling Otx1 + Otx2 with ab21990 at 1/300 dilution. The tissue was fixed with paraformaldehyde and permeabilized with PBS / 0.5% v/v Triton X-100. An undiluted donkey anti-rabbit Alexa Fluor® 555 secondary antibody was used.

    See Abreview

  • Anti-Otx1 + Otx2 antibody - ChIP Grade (ab21990) at 1 µg/ml + OTX1 (Human) - Recombinant Protein at 0.1 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 31.6 kDa
    Observed band size: 74 kDa
    why is the actual band size different from the predicted?



    This Otx1 recombinant protein has a GST tag causing it to run at ~74kDa.
  • ab21990 staining Otx2 in embryonic day 14.5 (E14.5) mouse eye by Immunohistochemistry (formalin fixed, paraffin embedded sections).
    The embryo was fixed in 10% neutral buffered formalin overnight at room temperature, then paraffin-embedded and sectioned at 4µm. Following deparaffinization and antigen retrieval in a rice steamer in 10mM sodium citrate (pH 6.0), the sections were blocked with 5% normal goat serum for half an hour at room temperature. ab21990 was diluted in PBS at 1/100, and incubated overnight at 4°C. The secondary was goat anti-rabbit Alexa®568, diluted in PBS with 1.5% normal goat serum. The mounting medium contained the nuclear stain DAPI (blue).

  • Mouse E6 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 ab21990 (1/2500) diluted in 10% serum, 1% ovalbumin in PBSTween for 14 hours at 4°C. They were then washed and stained with a goat anti-rabbit biotin conjugated antibody. After using a biotinylated secondary, ABC and then DAB was used for color development.

  • All lanes : Anti-Otx1 + Otx2 antibody - ChIP Grade (ab21990) at 1 µg/ml

    Lane 1 : Y79 Lysate
    Lane 2 : Y79 Lysate with Otx2 peptide (ab24347) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution

    Predicted band size: 31.6 kDa
    Observed band size: 37 kDa why is the actual band size different from the predicted?
    Additional bands at: 28 kDa (possible cleavage fragment), 28 kDa (possible isoform), 28 kDa (possible non-specific binding)

  • The image shows staining of Otx2 in human embryonic stem cells differentiated into neuroectoderm. As would be expected, staining is nuclear.

References

This product has been referenced in:
  • Aldunate EZ  et al. Conditional Dicer1 depletion using Chrnb4-Cre leads to cone cell death and impaired photopic vision. Sci Rep 9:2314 (2019). Read more (PubMed: 30783126) »
  • Paik EJ  et al. Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture. Sci Rep 8:804 (2018). Read more (PubMed: 29339826) »
See all 49 Publications for this product

Customer reviews and Q&As

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1-9 of 9 Abreviews

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293T)
Permeabilization
Yes - 0.1% Triton X-100
Specification
HEK293T
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 28 2017

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Zebra finch Tissue sections (brain)
Antigen retrieval step
None
Permeabilization
No
Specification
brain
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 10 2016

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing (10% SDS PAGE)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 02 2014

Application
Immunohistochemistry (Frozen sections)
Blocking step
NOVOCASTRA protein block as blocking agent as blocking agent as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 25°C
Sample
Mouse Tissue sections (E8.5 mouse embryo brain section)
Specification
E8.5 mouse embryo brain section
Permeabilization
Yes - PBS / 0.5% v/v Triton X-100 (Sigma)
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 05 2013

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Embryonic Stem Cells)
Specification
Embryonic Stem Cells
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton-X
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 29 2012

Application
Immunohistochemistry (Frozen sections)
Sample
Fish Tissue sections (Medaka and zebrafish eye and brain)
Specification
Medaka and zebrafish eye and brain
Fixative
Formaldehyde
Permeabilization
Yes - Triton x-100 0.2%
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Raquel Marco Ferreres

Verified customer

Submitted Jan 31 2011

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (E6.0 embryo in decidua)
Specification
E6.0 embryo in decidua
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
Yes - tween
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C

Dr. Jesse Mager

Verified customer

Submitted Dec 22 2008

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (E12.5 Whole brain section)
Specification
E12.5 Whole brain section
Fixative
Paraformaldehyde
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5%

Dr. Dean Griffiths

Verified customer

Submitted Jan 04 2007

Application
Western blot
Sample
Mouse Cell lysate - nuclear (GnRH neurons)
Loading amount
20 µg
Specification
GnRH neurons
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 2

Abcam user community

Verified customer

Submitted Jun 01 2006

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