RPE65 Antibody (401.8B11.3D9)

Images

 
Immunohistochemistry-Frozen: RPE65 Antibody (401.8B11.3D9) [NB100-355] - Staining of RPE65 in a cryosection of mouse retina tissue using RPE65 antibody (clone 401.8B11.3D9).
Western Blot: RPE65 Antibody (401.8B11.3D9) [NB100-355] - WB analysis of RPE65 in 20ug lysate of COS7 cells expressing recombinant Human RPE65 (Lane 1) and 5ug of Bovine retinal pigment epithelium membrane fraction ...read more
Immunocytochemistry/ Immunofluorescence: RPE65 Antibody (401.8B11.3D9) [NB100-355] - ICC-IF analysis of cultured ARPE19 cells, a spontaneously arising human retinal pigment epithelia cell line - 10 minutes fixation in ...read more
Immunohistochemistry: RPE65 Antibody (401.8B11.3D9) [NB100-355] - Zebrafish brain ventricular area labeled with mouse anti-RPE65 (1:100). This image was submitted through a verified customer review.
Western Blot: RPE65 Antibody (401.8B11.3D9) [NB100-355] - Subcellular localization of BEST1 and surface Ca2+-dependent Cl- current in patient-derived iPSC-RPEs. Western blots show similar BEST1 expression levels in WT ...read more
Immunocytochemistry/ Immunofluorescence: RPE65 Antibody (401.8B11.3D9) [NB100-355] - Expression of eye-specific markers in the induced eye-like structures induced from lignin-added ES cells. (a) Higher-magnification ...read more
Immunohistochemistry-Paraffin: RPE65 Antibody (401.8B11.3D9) [NB100-355] - Analysis of FFPE human glioblastoma tissue section using 1:500 dilution of RPE65 [401.8B11.3D9] antibody on a Bond Rx autostainer (Leica ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, Bv, Ca, Ch, Pm, XpSpecies Glossary
Applications WB, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, In vivo, CyTOF-ready, ICC/IF
Clone
401.8B11.3D9
Clonality
Monoclonal
Host
Mouse
Conjugate
Unconjugated
Concentration
1.0 mg/ml

RPE65 Antibody (401.8B11.3D9) Summary

Immunogen
This RPE65 Antibody (401.8B11.3D9) was developed against bovine RPE65 microsomal membrane proteins. [UniProt# Q28175]
Localization
Cytoplasmic, Microsomeal and Cell Membrane
Marker
Retinal Pigment Epithelium Marker
Isotype
IgG1 Kappa
Clonality
Monoclonal
Host
Mouse
Gene
RPE65
Purity
Protein G purified
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • Western Blot 1 - 2 ug/ml
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 1:50 - 1:200
  • Immunohistochemistry 1:250
  • Immunohistochemistry-Frozen 1:250
  • Immunohistochemistry-Paraffin 1:250-1:500
  • In vivo assay
  • CyTOF-ready
  • Immunofluorescence 1:50 - 1:200
Application Notes
For Western blot, this antibody has been validated in lysates of bovine RPE membrane and COS7 cells transfected with human RPE65, and in both samples, the antibody recognized a band at ~65 kDa, representing RPE65 protein. Note:- Hamel et al. have reported that in cultured bovine RPE cells, the levels of RPE65 are undetectable in WB after day 14, however, the mRNA levels are detectable by Northern for at least 7 weeks (PMID: 8340400). The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors. This antibody is CyTOF ready. Use in Immunoprecipitation reported by customer review. Use in In vivo reported in scientific literature (PMID:32173468).
Theoretical MW
65 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
Human Eye Tissue Lysate (Normal)
Mouse Eye Tissue Lysate (Normal)
Reviewed Applications
Read 7 Reviews rated 3.4
using
NB100-355 in the following applications:

Publications
Read Publications using
NB100-355 in the following applications:

Reactivity Notes

Primate reactivity reported in scientific literature (PMID: 31660416).

Packaging, Storage & Formulations

Storage
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Buffer
PBS
Preservative
0.02% Sodium Azide
Concentration
1.0 mg/ml
Purity
Protein G purified

Alternate Names for RPE65 Antibody (401.8B11.3D9)

  • All-trans-retinyl-palmitate hydrolase
  • BCO family, member 3
  • BCO3
  • EC 3.1.1.64
  • EC:3.1.1.64
  • EC:5.3.3.22
  • LCA2
  • lutein isomerase
  • meso-zeaxanthin isomerase
  • mRPE65
  • p63
  • RBP-binding membrane protein
  • rd12
  • retinal pigment epithelium specific protein 65
  • Retinal pigment epithelium-specific 65 kDa protein
  • retinal pigment epithelium-specific protein 65kDa
  • retinitis pigmentosa 20 (autosomal recessive)
  • retinoid isomerohydrolase
  • Retinol isomerase
  • RP20
  • RPE65
  • RPE65, retinoid isomerohydrolase
  • sRPE65

Background

RPE65 (retinal pigment epithelium-specific 65 kDa protein) was first described in 1991 and is a key isomerase in RPE that is largely localized to the smooth endoplasmic reticulum (1,2). The RPE65 gene is located on chromosome 1p31 and the protein is synthesized as 533 amino acids in length with a theoretical molecular weight of 61 kDa, although appearing near 65 kDa in SDS-PAGE (1,3). RPE65 serves an important enzymatic function in the visual cycle in converting all-trans-retinyl esters into 11-cis-retinal (1-6). In the visual cycle, light activates rhodopsin and other visual pigments in the photoreceptor cells (rods and cones), eventually generating all-trans-retinol which is transported to the RPE and converted to the vitamin A-derived chromophore 11-cis-rentinal via RPE65 and is then diffused back to the photoreceptor to continue the cycle (1-6). Although it is still unclear, some studies have suggested that S-palmitoylation post-translational modification of RPE65 is responsible for the stability and anchoring to the RPE membrane and therefore functioning enzymatic activity (1,2).

Given its essential role in the vision cycle, it is understandable that mutations in RPE65 are associated with a variety of inherited retinal dystrophies (1, 3-6). Leber Congenital Amaurosis (LCA) and retinitis pigmentosa (RP) are two of the most common retinal dystrophies associated with bi-allelic RPE65 gene mutations (5,6). In 2017 the FDA approved an in vivo gene therapy for treatment of RPE65-associated diseases (5,6). The drug Voretigene Neparvovec, also called Luxturna, is delivered sub-retinally and transduces RPE cells with cDNA encoding for normal RPE65 to help restore vision (5,6). There are several promising completed and ongoing clinical trials for treating RPE65-associated diseases using gene replacement therapy (5).

References

1. Kiser, P. D., & Palczewski, K. (2010). Membrane-binding and enzymatic properties of RPE65. Progress in retinal and eye research. https://doi.org/10.1016/j.preteyeres.2010.03.002

2. Uppal, S., Poliakov, E., Gentleman, S., & Redmond, T. M. (2019). RPE65 Palmitoylation: A Tale of Lipid Posttranslational Modification. Advances in experimental medicine and biology. https://doi.org/10.1007/978-3-030-27378-1_88

3. Redmond T. M. (2009). Focus on Molecules: RPE65, the visual cycle retinol isomerase. Experimental eye research. https://doi.org/10.1016/j.exer.2008.07.015

4. Saari J. C. (2016). Vitamin A and Vision. Sub-cellular biochemistry. https://doi.org/10.1007/978-94-024-0945-1_9

5. Miraldi Utz, V., Coussa, R. G., Antaki, F., & Traboulsi, E. I. (2018). Gene therapy for RPE65-related retinal disease. Ophthalmic genetics. https://doi.org/10.1080/13816810.2018.1533027

6. Apte R. S. (2018). Gene Therapy for Retinal Degeneration. Cell. https://doi.org/10.1016/j.cell.2018.03.021

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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NB100-355
Species: Hu, Mu, Rt, Po, Bv, Ca, Ch, Pm, Xp
Applications: WB, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, In vivo, CyTOF-ready, ICC/IF

Publications for RPE65 Antibody (NB100-355)(69)

We have publications tested in 8 confirmed species: Human, Mouse, Rat, Canine, Chicken, Mouse , Porcine, Primate.

We have publications tested in 7 applications: FLOW, ICC/IF, IHC, IHC-Fr, IHC-P, In vivo, WB.


Filter By Application
FLOW
(1)
ICC/IF
(24)
IHC
(15)
IHC-Fr
(10)
IHC-P
(4)
In vivo
(1)
WB
(10)
All Applications
Filter By Species
Human
(20)
Mouse
(21)
Rat
(5)
Canine
(6)
Chicken
(2)
Mouse
(1)
Porcine
(4)
Primate
(1)
All Species
Showing Publications 1 - 10 of 69. Show All 69 Publications.
Publications using NB100-355 Applications Species
Lee-Rivera I, Lopez E, Alvarez-Arce A, Lopez-Colome AM The PKC-zetz pseudosubstrate peptide induces glutamate release from retinal pigment epithelium cells through kinase- independent activation of Best1 Life sciences Dec 8 2020 [PMID: 33301813] (ICC/IF, Rat) ICC/IF Rat
Mitrousis N, Hacibekiroglu S, Ho M et al. Hydrogel-mediated co-transplantation of retinal pigmented epithelium and photoreceptors restores vision in an animal model of advanced retinal degeneration Biomaterials Jul 1 2020 [PMID: 32791386] (IHC, Mouse) IHC Mouse
Song J, Han D, Lee H et al. A Comprehensive Proteomic and Phosphoproteomic Analysis of Retinal Pigment Epithelium Reveals Multiple Pathway Alterations in Response to the Inflammatory Stimuli Int J Mol Sci Apr 25 2020 [PMID: 32344885] (ICC/IF, Human) ICC/IF Human
Ji C, Li Y, Kittredge A, et al. Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations Sci Rep Dec 13 2019 [PMID: 31836750] (WB, Human) WB Human
Shani S The implication of cell-derived microvesicles in retinal pigment epithelium degeneration Thesis Jul 11 1905 (IHC, Human) The implication of cell-derived microvesicles in retinal pigment epithelium degeneration. Thesis 2019 (IHC, Human)

Details:
Citation using the Alexa Fluor 488 version of this antibody.
IHC Human
Lehmann GL, Hanke-Gogokhia C, Hu Y et al. Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid J. Exp. Med. Jun 1 2020 [PMID: 32196081] (IHC-Fr, Mouse) IHC-Fr Mouse
Yang C, Shani S, Tahiri H et Al. Extracellular microparticles exacerbate oxidative damage to retinal pigment epithelial cells Exp. Cell Res. Mar 13 2020 [PMID: 32173468] (IHC, In vivo, Mouse) IHC, In vivo Mouse
Wright CB, Uehara H, Kim Y et al. Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice Proc. Natl. Acad. Sci. U.S.A. Jan 21 2020 [PMID: 31964819] (WB, Mouse) WB Mouse
Smith JR, Ashander LM, Ma Y et al. Model Systems for Studying Mechanisms of Ocular Toxoplasmosis Methods Mol. Biol. Nov 24 2019 [PMID: 31758460]
Lopez E, Lee-Rivera I, Alvarez-Arce A, Lopez-ColomE AM Thrombin induces Ca2+-dependent glutamate release from RPE cells mediated by PLC/PKC and reverse Na+/Ca2+ exchange Mol. Vis. Oct 5 2019 [PMID: 31673221] (ICC/IF, Human) ICC/IF Human
Show All 69 Publications.

Reviews for RPE65 Antibody (NB100-355) (7) 3.47

Average Rating: 3.4
(Based on 7 reviews)
We have 7 reviews tested in 3 species: Human, Human and Bovine, Other.

Reviews using NB100-355:
Filter by Applications
IP
(1)
ICC
(1)
IF
(3)
IHC-Fr
(1)
WB
(1)
All Applications
Filter by Species
Human
(3)
Human and Bovine
(1)
Other
(3)
All Species
Images Ratings Applications Species Date Details
Immunoprecipitation RPE65 NB100-355
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4
reviewed by:
Anonymous
IP Human and Bovine 05/06/2019
View

Summary

ApplicationImmunoprecipitation
Sample TestedBovine RPE cell extracts,293T cell lysate (overexpressing RPE65)
SpeciesHuman and Bovine
LotN

Comments

CommentsWe applied this antibody to endogenous RPE65 from bovine RPE cell extracts and human RPE65 overexpressed in 293T cells. In both cases it produced robust immunoprecipitation and good western blot signal. Four stars because it does recognize a relatively strong nonspecific band around 40 kD in 293T cell lysates.

Cells lysed in 1% IGEPAL

IP: 2.5 uL RPE65 antibody per approximately 350 ug protein using A/G agarose beads

WB dilution: 1:1000
Immunocytochemistry RPE65 NB100-355
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3
reviewed by:
Fabio Marongiu
ICC Human 08/29/2017
View

Summary

ApplicationImmunocytochemistry
Sample TestedARPE-19 cells
SpeciesHuman
LotJ-1

Comments

CommentsFixative Methanol
Blocking Goat Serum
Dilution 1:250
Incubation 1h RT
Immunofluorescence RPE65 NB100-355
Enlarge
3
reviewed by:
Anonymous
IF Other 03/23/2016
View

Summary

ApplicationImmunofluorescence
Sample Testedpfa fixed 11day old zebrafish cryosections
SpeciesOther

Blocking

Blocking Details3hr with 20% goat serum in 0.1%Tween PBS

Primary Anitbody

Dilution Ratiobest 1:100 overnight at 4degrees (also tested 1:250, 1:500)

Secondary Antibody

Secondary Descriptionlife technology Goat anti- mouse Alexa 546 (1:200)
Secondary Manufacturer Cat#life technologies (Molecular Probes)
Secondary Concentration1:200

Details

Detection Notesconfocal (negative control, no primary and serial dilution)
Fixation Details1 hour 4% pfa fix at room temp, cryoprotected, sectioned to 25um
Wash Description2x PBS 1 hour

Comments

Commentsthis antibody does not recognize RPE65a in the RPE in zebrafish (no labeling in eye). Probably recognizing RPE65b.
  4
reviewed by:
Anonymous
IHC-Fr Other 12/12/2014
View

Summary

ApplicationImmunohistochemistry-Frozen
Sample TestedSee PMID 24143172
SpeciesOther
  4
reviewed by:
Girish Kumar Srivastava
IF Other 10/06/2014
View

Summary

ApplicationImmunofluorescence
SpeciesOther
Pub Med ID24719852
FileView PDF
Immunofluorescence RPE65 NB100-355
Enlarge
4
reviewed by:
Girish Kumar Srivastava
IF Human 04/30/2014
View

Summary

ApplicationImmunofluorescence
SpeciesHuman
Pub Med ID21442725
FileView PDF
  2
reviewed by:
Anonymous
WB Human 12/06/2010
View

Summary

ApplicationWestern Blot
Sample TestedRPE whole cell lysate, Sample Amount: 20-25microgram
SpeciesHuman
CommentsUsing RPE cell lysates, I can detect RPE65 protein by western blot analysis. However, the antibody works with high cross-reactivity and background. In addition, a band at 65kDa, appears as a doublet instead of a single band. The data sheet is showing a single band at 65kDa. Therefore, I am not sure why the results are unclear!

Blocking

Blocking DetailsBlocking Buffer: 2.5% Milk in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Dilution Buffer: 5% BSA in 1X TBST, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab: anti-mouse HRP (Sigma), Secondary Ab Dilution Ratio: 1:8000
Secondary Manufacturer Cat#A3682

Details

Detection NotesDetection Method: Chemiluminescence, Exposure Time: 30sec-1min, Specific Bands: 65 kDa, Non-Specific Bands: 40-45kDa

Comments

CommentsUsing RPE cell lysates, I can detect RPE65 protein by western blot analysis. However, the antibody works with high cross-reactivity and background. In addition, a band at 65kDa, appears as a doublet instead of a single band. The data sheet is showing a single band at 65kDa. Therefore, I am not sure why the results are unclear!

Product General Protocols

View specific protocols for RPE65 Antibody (NB100-355): Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

WB Video Protocol
ICC/IF Video Protocol

FAQs for RPE65 Antibody (NB100-355). (Showing 1 - 5 of 5 FAQs).

  1. I have one question regarding the immunoblot of the RPE65 I always got two bands in positive control. I am not able to understand why. Please clarify my doubt. I am unsing Novus RPE65 antibody.
    • RPE65 is subject to several different post-translational modifications that influence its apparent band size in an SDS-PAGE gel. Please see UniProt to learn more. The doublet you are seeing (which we also observe) most likely reflects an unmodified and a modified version of the protein. I hope this helps, but please let me know if you have additional questions.
  2. I am thinking about buying this ab (RPE65 Antibody); you have a nice picture showing how it is working in a saggital retinal section. Could I have the protocol that was used in this labeling? Frozen sections? Antigen retrieval?
    • Thank you for your email! We do not have a specific protocol for this Ab. Please see the Immunohistochemistry-Frozen Protocol our lab follows. The recommended dilution for IHC-Fr is 1:250 and antigen retrieval is not neccessary as the the protein is located in the cytoplasm.
  3. We have a question regarding species-specificity for this antibody. Does it work on rat? We'd like to use it on rat eye cryosections. But on the data sheet, only Bovine, Human, Mouse and Porcine were listed.
    • We have not tested RPE65 on rat samples. There is 93% similarity between the rat and bovine proteins, which is very promising but I still cannot guarantee that it will work. If you would like to test this antibody in rat samples, you would qualify for the Innovator's Reward Program which rewards our customers for trying our antibodies in previously untested species and applications.
  4. For clarification, is the 1:250 the recommended dilution for only IHC-F or all applications preceding IHC-F on the data sheet? If not, is there a recommended dilution for IHC-P? Do you have any information on antigen retrieval? Citrate buffer or higher pH (EDTA)? Temperature? Time? Regarding incubation time, is 1 hour at room temperature or overnight at 4C recommended?
    • The dilution of 1:250 should suffice for IHC-P as well as frozen sections. Unfortunately this item was tested by an outside collaborator who did not provide us with the protocol they used. Generally we recommend using antigen retrieval with citrate buffer and incubating for 1 hour at RT. Some antigens may be tricky and require overnight incubation. As all samples are different, this usually has to be user determined.
  5. Do you recommend an antigen retrieval step for this antibody : RPE65 Antibody (401.8B11.3D9)?. My application is IHC-paraffin cross section!
    • Yes, we do recommend antigen retrieval step for NB100-355 and in our IHC validation for this product, heat induced method was employed. You may use citrate buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0) for this purpose. Please view more information on antigen retrieval protocols.

Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 405 NB100-355AF405
Alexa Fluor 488 NB100-355AF488
Alexa Fluor 647 NB100-355AF647
Alexa Fluor 700 NB100-355AF700
Allophycocyanin NB100-355APC
Biotin NB100-355B
DyLight 350 NB100-355UV
DyLight 405 NB100-355V
DyLight 488 NB100-355G
DyLight 550 NB100-355R
DyLight 650 NB100-355C
DyLight 680 NB100-355FR
DyLight 755 NB100-355IR
FITC NB100-355F
HRP NB100-355H
PE NB100-355PE
PerCP NB100-355PCP

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Array NB100-355

Bioinformatics Tool for RPE65 Antibody (NB100-355)

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Blogs on RPE65.

Using RPE65 as a tool to investigate ocular gene therapies
While not life threatening, blindness and retinal disease are profoundly debilitating and greatly affect quality of life.  Understandably, gene therapy has been subject to controversy given it’s potential effects on the rest of our cellular...  Read full blog post.

RPE65: Vision, Blindness and Hope
Retinal pigment epithelium-specific 65 kDa protein (RPE65) is an essential vision protein, and so mutations in the RPE65 gene cause blindness. However, clinical trials using gene therapy to treat patients with a defective RPE65 gene suggest that some...  Read full blog post.

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Recent Reviews

3.4
5
0
4
4
3
2
2
1
1
0

Anonymous
05/06/2019
Application: IP
Species: Human and Bovine

Fabio Marongiu
08/29/2017
Application: ICC
Species: Human

Anonymous
03/23/2016
Application: IF
Species: Other

Bioinformatics

Gene Symbol RPE65