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

1-10 of 10 Q&A

Answer

Thank you for your enquiry. The location of the data is not obvious. The ab21990 ChIP data in PubMed:18057103 is in the supplemental material for this paper. Here is a link:

http://dev.biologists.org/content/135/2/249/suppl/DC1

Chromatin samples were prepared from heads of uninjected stage 22-24 embryos, and imunoprecipitated with either 5 micrograms of control IgG or anti-Otx2 antibody (Abcam, ab21990).

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Answer

Thank you for contacting Abcam regarding ab21990.


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.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you for contacting us.
I'm sorry to hear that the customer has trouble with this antibody.

The information that was provided by the customer is very helpful to us and I am happy to arrange for a free of charge replacement with ab21990. It will be added to our next shipment to you.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. 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 with the order number . To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.

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Answer

Thank you for getting back to me with these questions.

I have been under the assumption that you havwe been using ab21190 for your experiments, have you been using ab92515 as well?Based on sequence homology ab92515 will recognize both proteins. The absence of a second band in the image is unresolved but may be due to poor separation or extrememly low levels of Otx1 in the sample.

Using ab21190 the lower band will be Otx2 which, in either isoform, has a lower molecular weight that Otx1. (http://www.uniprot.org/uniprot/P32243&http://www.uniprot.org/uniprot/P32242).

Although ab76748 does not have any western blot images in the datasheet we have been assured that the immunogen used for this has no cross reactivity to Otx1.This product is guaranteed to work in western bloton ​human tissues and is covered by our Abpromise for a period of six months. Since we have been unable to resolve this problem, if you have purchased this product within the last 180 days, I would be happy to either send this replacement antibody or to process a refund. If you could please supply me with your Abcam order confirmation number for the purchase of ab21190 I can begin to process this replacement,

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Answer

Thank you for contacting us.

The immunogen for ab92515 shares 100% identity with OTX1, so cross reactivity is predicted.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for contacting us.

I am sorry that you have found this product unsatisfactory following its recent target refinement. As we discussed on the phone the sequence for ab21990 is shared by both OTX1 and OTX2. It was because of this that we have changed the targets.

We have other products which has been guaranteed to work in WB and ICH on human tissue. One such product is ab76748. According to information that I have recieved form the lab, this antibody is specific to Otx2 only with no cross reactivity with Otx1. The immunogenic peptide was selected from specific domains of Otx2.


Another product I would like to mention is ab92326. The immunogen for this OTX2 antibody shares 81% identity with OTX1, so the antibody is not predicted to cross-react with OTX1. However, it is not guaranteed without testing

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us. We have shipped lot GR9703-1 in your order last time; we still have this lot in stock and our retesting results are quite promising. I am sorry that the product failed in your experiment. I would like to know more about the experiment you have performed. Could you provide the following information? - Which application the antibody was used in? - How do you describe the problem? -Could you attach a brief protocol and any information regarding troubleshooting steps? I will look forward to hearing from you soon.

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Answer

Thank you for contacting us. The affected orders was 030Z001155. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us. The lot number you have received was 831530. This lot is also affected by the specificity data we currently got for this product. This product should have detected OTX1 and OTX2 both. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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