Overview

  • Product name
    Anti-RPE65 antibody [401.8B11.3D9]
    See all RPE65 primary antibodies
  • Description
    Mouse monoclonal [401.8B11.3D9] to RPE65
  • Host species
    Mouse
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Fr, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Pig
    Predicted to work with: Dog
  • Immunogen

    Tissue, cells or virus corresponding to RPE65. Bovine RPE microsomal membrane proteins.

  • Positive control
    • WB: Recombinant human RPE protein expressed in COS-7 cells and bovine RPE membrane fraction. IHC-Fr: Mouse retina tissue. ICC/IF: ARPE19 cells. Flow Cytometry: Mouse retina and choroid cells.
  • General notes

    Expression in primary culture of RPE cells is undetectable in Western blot after day 14. Established cell lines are similarly devoid of RPE65 protein, as indicated in western blot. However, mRNA levels are detectable by Northern for at least 7 weeks. (see reference Hamel, C.P., et al. Molecular Cloning and expression of RPE65 a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. JBC. 268(21): 15751-15757, 1993.)

     

Properties

Applications

Our Abpromise guarantee covers the use of ab13826 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
ICC/IF 1/1000. PubMed: 170320581/1000 (from PubMed:17032058).
WB 1/5000 - 1/10000. Detects a band of approximately 65 kDa.
IHC-Fr 1/250.
Flow Cyt Use at an assay dependent concentration. PubMed: 21494877

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Plays important roles in the production of 11-cis retinal and in visual pigment regeneration. The soluble form binds vitamin A (all-trans-retinol), making it available for LRAT processing to all-trans-retinyl ester. The membrane form, palmitoylated by LRAT, binds all-trans-retinyl esters, making them available for IMH (isomerohydrolase) processing to all-cis-retinol. The soluble form is regenerated by transferring its palmitoyl groups onto 11-cis-retinol, a reaction catalyzed by LRAT. The enzymatic activity is linearly dependent of the expression levels and membrane association.
  • Tissue specificity
    Retinal pigment epithelium specific.
  • Involvement in disease
    Defects in RPE65 are the cause of Leber congenital amaurosis type 2 (LCA2) [MIM:204100]. LCA designates a clinically and genetically heterogeneous group of childhood retinal degenerations, generally inherited in an autosomal recessive manner. Affected infants have little or no retinal photoreceptor function as tested by electroretinography. LCA represents the most common genetic cause of congenital visual impairment in infants and children.
    Defects in RPE65 are the cause of retinitis pigmentosa type 20 (RP20) [MIM:613794]. RP leads to degeneration of retinal photoreceptor cells. Patients typically have night vision blindness and loss of midperipheral visual field. As their condition progresses, they lose their far peripheral visual field and eventually central vision as well. RP20 inheritance is autosomal dominant.
  • Sequence similarities
    Belongs to the carotenoid oxygenase family.
  • Post-translational
    modifications
    Palmitoylation by LRAT regulates ligand binding specificity; the palmitoylated form (membrane form) specifically binds all-trans-retinyl-palmitate, while the soluble unpalmitoylated form binds all-trans-retinol (vitamin A).
  • Cellular localization
    Cytoplasm. Cell membrane. Attached to the membrane by a lipid anchor when palmitoylated (membrane form), soluble when unpalmitoylated.
  • Information by UniProt
  • Database links
  • Alternative names
    • All-trans-retinyl-palmitate hydrolase antibody
    • LCA 2 antibody
    • LCA2 antibody
    • Leber congenital amaurosis antibody
    • mRPE 65 antibody
    • mRPE65 antibody
    • p63 antibody
    • rd 12 antibody
    • rd12 antibody
    • Retinal pigment epithelium specific 61 kDa protein antibody
    • Retinal pigment epithelium specific 65 kDa protein antibody
    • Retinal pigment epithelium specific protein antibody
    • Retinal pigment epithelium specific protein 65kDa antibody
    • Retinal pigment epithelium-specific 65 kDa protein antibody
    • Retinitis pigmentosa 20 antibody
    • Retinoid isomerohydrolase antibody
    • Retinol isomerase antibody
    • RP 20 antibody
    • RP20 antibody
    • RPE 65 antibody
    • RPE65 antibody
    • RPE65_HUMAN antibody
    • sRPE 65 antibody
    • sRPE65 antibody
    see all

Images

  • All lanes : Anti-RPE65 antibody [401.8B11.3D9] (ab13826) at 1/5000 dilution

    Lane 1 : Recombinant human RPE-transfected COS-7 (African green monkey kidney fibroblast-like cell line) cell lysate at 20 µg
    Lane 2 : Bovine retinal pigment epithelium membrane fraction at 5 µg

    Secondary
    All lanes : Alkaline phosphatase conjugated goat-anti Mouse IgG

    Observed band size: 60 kDa
    why is the actual band size different from the predicted?



    NBT/BCIP substrate

  • ARPE19 (spontaneously arising human retinal pigment epithelia cell line) cells stained for RPE65 (green) using ab13826 (1/100 dilution) in ICC/IF.

    Cells were fixed in 4% PFA for 10 minutes, permeabilized in PBS containing 0.2% Triton X-100 (PBS-T) for 10 minutes, then blocked for 1 hour in 10% normal goat serum containing 1% BSA in PBS-T. Incubated with primary antibody at 4°C.

  • Frozen sectioned mouse retina tissue stained for RPE65 with ab13826 at a 1/250 dilution in immunohistochemical analysis.

  • Flow Cytometry analysis of mouse retina and choroid cells stained for RPE65 using ab13826.

    RPE65 (right) versus control (left) in the choroid (top) and the retina (bottom).

References

This product has been referenced in:
  • Kelley RA  et al. DNA nanoparticles are safe and nontoxic in non-human primate eyes. Int J Nanomedicine 13:1361-1379 (2018). Read more (PubMed: 29563793) »
  • Wang J  et al. ATAC-Seq analysis reveals a widespread decrease of chromatin accessibility in age-related macular degeneration. Nat Commun 9:1364 (2018). Flow Cyt . Read more (PubMed: 29636475) »
See all 34 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
Answer

Merci de nous avoir contactés.

L'anticorps 13826 est un monoclonal, il ne reconnait donc pas toute la séquence mais uniquement une région très précise de l'immunogène qui a été utilisé (toute la protéine de RPE bovin). Malheureusement cet épitope (qui correspond en règle générale à +/- 8 acides aminés) n'a pas été déterminé.

Nous avons vérifié la similarité entre la séquence d'acides aminés du rat et de la vache. Il est très probable que l’anticorps fonctionne chez le rat (voir la piece jointe).

Comme nous avons plus de 90.000 références, nous ne pouvons stocker différents volumes pour nos produits et nous n'offrons donc pas d'échantillon gratuit ou de petit volume pour des essais préliminaires.

Notre politique est que si un anticorps ne marche pas comme spécifié dans sa fiche technique, nous offrons un remboursement ou un remplacement gratuit (Garantie Abpromise, www.abcam.com/abpromise).

Si vous souhaitez utiliser cet anticorps contre le rat, nous pouvons vous offrir une offre de test :

1. Nous informer que vous souhaitez tester un de nos anticorps avec une espèce ou dans une application encore non-testée et nous vous enverrons un code promotionnel (valable 4 mois) à utiliser après obtention et envoi de vos résultats,

2. Acheter l'anticorps,

3. Nous envoyer vos résultats (positifs ou négatifs) obtenus avec une espèce ou dans une application encore non testée sous forme d'Abreview (www.abcam.com/abreviews). Ceci activera le code.

4. Commander gratuitement un anticorps primaire de notre catalogue.

J'espère que ces informations vous aideront. N'hésitez pas à me contacter si vous avez besoin de plus de renseignements.

Read More

Answer

Merci de votre réponse.

Nous n'avons pas un autre lot de l'ab13826 en stock. Comme vous l'avez souhaité, je vous ai envoyé donc une unité de l'ab78036. Je me permets de vous transférer le numéro de commande de remplacement gratuit de ce produit :... Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

Sachez que aussi le tube de remplacement est garantie par notre Abpromise https://www.abcam.com/abpromise

N'hésitez pas à nous contacter si vous avez des questions.

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Answer

Merci d'avoir pris le temps de mettre ensemble ce dossier et de nous avoir contactés. Je suis désolée d'apprendre que vous avez eu des difficultés à obtenir de bons résultats avec ab13826.

Après étude de votre protocole, je pense que cet anticorps aurait du fonctionner et jedois conclure que il semble que vous avez reçu un tube fautif malheureusement. Je serais ravis de vous envoyer soit un tube de remplacement de cet anticorps ou un anticorps alternative, soit de vous organiser un avoir/remboursement. Veuillez s'il vous plaît me confirmer comment vous souhaitez procéder.

Pouvez vous s'il vous plaît aussi me confirmer le numéro de commande original de l'ab13826 ou sinon la date de la commande avec le nom de la personne qui avait passé la commande? Ceci va me permettre d'identifier le tube et d'organiser le remplacement.

Merci de votre coopération. Je me réjouis de votre réponse.

Read More

Answer

Thank you for your inquiry and your patience while we were consulting with the laboratory. I am sorry to hear you are experiencing difficulties with one of our products.

Unfortunately we do not know whether this antibody does react with proteins expressed on mouse fibroblasts cells or cell lines and we have not tested this antibody on mouse fibroblasts neither.

Unfortunately, theepitope of this antibody has not been mapped by us and we are not aware if anyone elsedid the epitope mapping.

I am sorry also that the laboratory has no other images to send. The image is not magnified enough to see if the staining is punctuate, and one can only see that it is staining cytoplasmic in the epithelium.

I would like however to reassure you that we do guarantee all ourantibodies to work as stated on the datasheet. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (RPE cells extracted from mouse eyes)
Loading amount
25 µg
Specification
RPE cells extracted from mouse eyes
Gel Running Conditions
Reduced Denaturing (10)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Nov 25 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (mouse eye sections)
Specification
mouse eye sections
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% TX100
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: 25°C

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Nov 25 2010

Answer

I'm afraid the epitope recognized by ab13826 has not been mapped yet so we do not know if the antibody is likely to cross react with Xenopus protein. The immunogen used to generate the antibody was whole Bovine RPE microsomal membrane proteins therefore the epitope could be anywhere on the protein. I'm sorry I couldn't help you more.

Read More

Answer

I'm sorry to hear you are having a problem with ab13826 in IHC, we have not tested this antibody in IHC or ICC so it is difficult to find out what might be the problem, the fact that the antibody is a mouse monoclonal and you use it on mouse tissue is a very likely one. There are indeed means to "tag" primary antibodies with fluorescence ; those are commercially available kits, you may find the website biocompare.com useful to find those. Furthermore it is possible that the background is due to the primary antibody not recognising the native epitope or that certain steps in your protocol promote the background. I hope this information helps, please do not hesitate to contact us for further advice,

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