Recombinant
RabMAb

Recombinant Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

Submit a review Q&A (4)References (1)

Overview

  • Product name

    Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade
    See all Otx1 + Otx2 primary antibodies

  • Description

    Rabbit monoclonal [EPR3347] to Otx1 + Otx2 - ChIP Grade

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, IP, ChIPmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    This information is considered to be commercially sensitive.

  • Positive control

    • WB: Y79 whole cell lysate and Otx1 recombinant protein. IHC-P: Human retina, human retinoblastoma, human cerebral cortex, mouse retina, mouse E14.5 choroid plexus and rat E14.5 choroid plexus. ChIP: Mouse stem cell D3. IP: Y79 whole cell lysate

  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab92515 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
WB 1/1000 - 1/10000. Predicted molecular weight: 32 kDa.
IHC-P 1/500 - 1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

We recommend 1/500 dilution for human and mouse tissues, and 1/1000 for rat tissues.
IP 1/20.
ChIP Use 5 µg for 25 µg of chromatin.

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

    see all

  • 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

  • Western blot - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Western blot - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1/2500 dilution (purified) + Y79 cell lysate at 10 µg

    Secondary
    HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

    Predicted band size: 32 kDa
    Observed band size: 32 kDa



    Blocking buffer: 5% NFDM/TBST
    Dilution buffer: 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human retina tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

    Nuclear staining on inner nuclear layer and outer nuclear layer of the human retina.

  • ChIP - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    ChIP - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Chromatin was prepared from mouse stem cell D3 according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab92515 (blue), and 20µl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

  • Immunoprecipitation - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunoprecipitation - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Otx1 + Otx2 was immunoprecipitated from Y79（Human retinoblastoma retinoblastoma）whole cell lysate with ab92515 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab92515 at 1/500 dilution. Secondary antibody details (ab131366), was used as secondary antibody at 1/1000 dilution.

    Blocking/Diluting Buffer and concentration: 5% NFDM /TBST.



    Lanes 1-2 : Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1/500 dilution (purified)
    Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab92515

    Lane 1 : Y79 (Human retinoblastoma retinoblastoma) whole cell lysate
    Lanes 2-3 : Y79 whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
  • Western blot - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Western blot - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515) at 1 µg/ml (unpurified) + OTX1 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: 32 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human retinoblastoma tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

    Nuclear staining on tumor cells of human retinoblastoma.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse retina tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

    Nuclear staining on nuclear layer of the mouse retina.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse E14.5 choroid plexus tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

    Nuclear staining on mouse E14.5 choroid plexus.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat E14.5 choroid plexus tissue labelling Otx1 + Otx2 with purified ab92515 at a dilution 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 (ab93684). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

    Nuclear staining on rat E14.5 choroid plexus.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Otx1 + Otx2 antibody [EPR3347] - ChIP Grade (ab92515)
    Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab92515 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

References

This product has been referenced in:

  • Saini JS  et al. Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration. Cell Stem Cell : (2017). Read more (PubMed: 28132833) »
See 1 Publication for this product

Publishing research using ab92515? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-4 of 4 Abreviews or Q&A

Question

Hi,
I bought anti Otx1/ Otx2 Ab (Ab92515) from your company. I could not find if it works on denatured and reduced protein or native in western?

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Answer

Thank you for contacting us.

Our antibodies are tested in WB under reducing and denaturing conditions unless specified otherwise on the datasheet. So for this antibody I would recomend reducing/denaturing conditions as it has not been tested in native.

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

I have only been using ab21990 the order confirmation number is .   Please go ahead and send me ab76748 as a replacement. Thank you for sending time on resolving these issues.  

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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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Question

Here is the link to ab92515 ( https://www.abcam.com/Otx2-antibody-EPR3347-ab92515.html) The western has a single band close to the 25kD marker band.    While the ab21990 has two bands one just above 25kD and one at 37kD. Since 21990 detects both OTX1 (37kD) and OTX2 (32kD) I assume my lower band is OTX2. But if ab92515 detects both proteins then why don’t we see two bands?   Whereas ab76748 that you suggested previously doesn’t have any western images in the datasheet.   I am trying to estimate amounts of OTX2 only. And this is important hence all the questions.  

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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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Question

How about ab92515? Is there a reason you have not mention this Antibody – except that it is monoclonal and the others polyclonal? Does the immunogen cross react ? It has images that seem to indicate specificity to OTX2 only,  in a western.  

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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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