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
Immunocytochemistry/ Immunofluorescence: RPE65 Antibody (401.8B11.3D9) [NB100-355] - ARPE-19 monolayer on glass substrate. Fixative Methanol Blocking Goat Serum. Dilution 1:250. Incubation 1h RT. This image was ...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
Immunofluorescence: 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.

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, Bv, Ca, Ch, XpSpecies Glossary
Applications WB, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, 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
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 1:500 - 1:1000
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 1:10-1:500
  • Immunohistochemistry 1:250
  • Immunohistochemistry-Frozen 1:250
  • Immunohistochemistry-Paraffin 1:250-1:500
  • CyTOF-ready
  • Immunofluorescence
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.
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

Based on immunogen sequence similarity, this antibody is predicted to react with most Birds species (89%), Zebrafish (74%) and Sea lamprey (71%).

Packaging, Storage & Formulations

Storage
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Buffer
PBS
Preservative
0.05% Sodium Azide
Concentration
1.0 mg/ml
Purity
Protein G purified

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

  • All-trans-retinyl-palmitate hydrolase
  • EC 3.1.1.64
  • EC 5.2.1.7
  • LCA2p63
  • mRPE65
  • RBP-binding membrane protein
  • rd12
  • Retinal pigment epithelium-specific 65 kDa protein
  • retinal pigment epithelium-specific protein (65kD)
  • retinal pigment epithelium-specific protein 65kDa
  • retinitis pigmentosa 20 (autosomal recessive)
  • retinoid isomerohydrolase
  • Retinol isomerase
  • RP20
  • sRPE65

Background

RPE65 (retinal pigment epithelium-specific 65 kDa protein), also called retinoid isomerohydrolase, is an essential retinoid isomerase enzyme of the canonical retinal pigment epithelium/RPE visual cycle and it is highly /preferentially expressed in the RPE layer, monolayer of neuroepithelium-derived cells located between the choroid and photoreceptors of the eye. It is responsible for retinol isomerization (essential reaction required for functional vision) and it converts all-trans retinyl ester to 11-cis retinol. RPE65 protein is functionally conserved throughout the vertebrates, and it is commonly used as an RPE marker by the research community. The loss of RPE65 function has been suggested to involve two distinct mechanisms - loss of catalytic activity and a lower expression level of RPE65. More than 80 different mutations have been reported in this protein and RPE65 gene mutations have been shown to be the cause of Leber's congenital amaurosis (LCA, MIM204000), a severe congenital or early infant-onset form of inherited retinal dystrophy.

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, Xp
Applications: WB, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, CyTOF-ready, ICC/IF

Publications for RPE65 Antibody (NB100-355)(53)

We have publications tested in 7 confirmed species: Human, Mouse, Rat, Canine, Chicken, Mouse , Porcine.

We have publications tested in 6 applications: FLOW, ICC/IF, IHC, IHC-Fr, IHC-P, WB.


Filter By Application
FLOW
(1)
ICC/IF
(21)
IHC
(9)
IHC-Fr
(8)
IHC-P
(4)
WB
(7)
All Applications
Filter By Species
Human
(15)
Mouse
(16)
Rat
(3)
Canine
(4)
Chicken
(2)
Mouse
(1)
Porcine
(4)
All Species
Showing Publications 1 - 10 of 53. Show All 53 Publications.
Publications using NB100-355 Applications Species
Hellinen L, Pirskanen L, Tengvall-Unadike U et al. Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties Pharmaceutics Aug 13 2019 [PMID: 31412689] (ICC/IF) ICC/IF
Scruggs BA, Jiao C, Cranston CM et al. Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments Stem Cells Transl Med Aug 1 2019 [PMID: 31004408] (IHC, Porcine) IHC Porcine
Cai, H;Gong, J;Abriola, L;Hoyer, D;Stem Cell Array Team, NG;Noggle, S;Paull, D;Del Priore, LV;Fields, MA; High-throughput screening identifies compounds that protect RPE cells from physiological stressors present in AMD Exp. Eye Res. Apr 10 2019 [PMID: 30980814] (ICC/IF, Human) ICC/IF Human
Cho IH, Park SJ, Lee SH et al. The role of Wnt/B-catenin signaling in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium after laser photocoagulation. Lasers Med Sci Sep 22 2018 [PMID: 30244402] (WB, Human) WB Human
Bhutto IA, Ogura S, Baldeosingh R et al. An Acute Injury Model for the Phenotypic Characteristics of Geographic Atrophy. Invest. Ophthalmol. Vis. Sci. Mar 20 2018 [PMID: 30208410] (IHC-Fr, Rat) IHC-Fr Rat
Kaur G, Tan LX, Rathnasamy G et al. Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration Proc. Natl. Acad. Sci. U.S.A. Aug 20 2018 [PMID: 30126999] (WB, Mouse) WB Mouse
Chae JB, Rho CR, Shin JA et al. Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells Korean J Ophthalmol Aug 1 2018 [PMID: 30091312] (WB, ICC/IF, Human) WB, ICC/IF Human
Gao Z, Liao Y, Chen C et al. Conversion of all-trans-retinal into all-trans-retinal dimer reflects an alternative metabolic/antidotal pathway of all-trans-retinal in the retina. J. Biol. Chem. Jul 26 2018 [PMID: 30049796]
Llonch S, Carido M, Ader M. Organoid technology for retinal repair. Dev. Biol. Jan 15 2018 [PMID: 29291970] (IHC, Mouse) IHC Mouse
Li Y, Zhang Y, Xu Y et al. Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca(2+)-dependent Cl(-) currents in human RPE. Elife. 2017 Oct 24 [PMID: 29063836] (WB, Human) WB Human
Show All 53 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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reviewed by:
Anonymous
IP Human and Bovine 05/06/2019
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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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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
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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.
 
reviewed by:
Anonymous
IHC-Fr Other 12/12/2014
View

Summary

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

Summary

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

Summary

ApplicationImmunofluorescence
SpeciesHuman
Pub Med ID21442725
FileView PDF
 
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

Additional RPE65 Products

Array NB100-355

Bioinformatics Tool for RPE65 Antibody (NB100-355)

Discover related pathways, diseases and genes to RPE65 Antibody (NB100-355). Need help? Read the Bioinformatics Tool Guide for instructions on using this tool.
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Diseases 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