RPE65 Antibody (401.8B11.3D9) - BSA Free

Western Blot: RPE65 Antibody (401.8B11.3D9) - BSA Free [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 and patient-derived iPSC-RPEs. Each sample was from one cell lysis (BEST1 and beta-actin, RPE65 and CRALBP were on two gels, respectively). Image collected and cropped by CiteAb from the following publication (//elifesciences.org/articles/29914), licensed under a CC-BY licence.

Immunocytochemistry/Immunofluorescence: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - Expression of eye-specific markers in the induced eye-like structures induced from lignin-added ES cells. (a) Higher-magnification image of the RPE like structure induced from ESCs after 12-day culture. (b) Immunostaining of eye- like structures. Eye- like structures induced from ESCs after 12-day culture were stained with antibodies against RPE65 (red) and nuclei were stained with DAPI solution (blue). Scale bar: a = 50 um, b,d = 200 um, c,e = 100 um. PLoS One. 2013 Jun 21;8(6):e66376. doi: 10.1371/journal.pone.0066376.

Immunohistochemistry-Paraffin: RPE65 Antibody (401.8B11.3D9) - BSA Free [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 Biosystems). The assay involved 20 minutes of heat induced antigen retrieval (HIER) with 10mM sodium citrate buffer (pH 6.0) and endogenous peroxidase quenching using peroxide block. The sections were incubated with primary antibody for 30 minutes. Bond Polymer Refine Detection (Leica Biosystems) and DAB were used for signal detection which followed counterstaining with hematoxylin. Whole slide scanning and capturing of representative images (20X) were performed using Aperio AT2 (Leica Biosystems).

Western Blot: RPE65 Antibody (401.8B11.3D9) - BSA Free [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 (Lane 2). Blot processed for detection with alkaline phosphatase conjugated goat-anti Mouse IgG secondary antibody and NBT/BCIP substrate.

Immunocytochemistry/Immunofluorescence: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - ICC-IF analysis of cultured ARPE19 cells, a spontaneously arising human retinal pigment epithelia cell line - 10 minutes fixation in 4% PFA, 10 minutes permeabilization in PBS containing 0.2% Triton X-100 (PBS-T), 1 hour blocking in 10% normal goat serum containing 1% BSA in PBS-T, 1:100 primary antibody dilution in PBS, ON 4C incubation.

Immunohistochemistry-Frozen: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - Staining of RPE65 in a cryosection of mouse retina tissue using RPE65 antibody (clone 401.8B11.3D9).

Immunohistochemistry: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - Zebrafish brain ventricular area labeled with mouse anti-RPE65 (1:100). This image was submitted through a verified customer review.

Simple Western: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - iPSC-RPE from all donors express RPE marker proteins. iPSC-RPE from the patient, the patients mom, the patients dad, and the three unrelated, unaffected controls all expressed RPE65. The negative control (iPSCs) had no detectable RPE65 protein expression. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/29540715/) licensed under a CC-BY licence.

Knockout Validated: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355] - Immunoblotting showing the expression of RPE-specific proteins BEST1 (NB300-164), RPE65 (NB100-355), CRALBP, and the loading control beta-Actin in iPSC-RPE cells. Two gels/blots in the same panel were prepared from the same cell lysate of each PSC-RPE to detect BEST1 + beta-Actin, and RPE65 + CRALBP, respectively. Image collected and cropped by CiteAb from the following publication (//pubmed.ncbi.nlm.nih.gov/34061021/) licensed under a CC-BY licence.

• Reactivity: Hu, Mu, Rt, Po, Bv, Ca, Ch, Pm, Xp
• Applications: WB, Simple Western, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, In vivo, CyTOF-ready, ICC/IF, KO
• Clone: 401.8B11.3D9
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

RPE65 Antibody (401.8B11.3D9) - BSA Free 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

Applications/Dilutions

• Dilutions:
  • CyTOF-ready
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 1:50 - 1:200
  • Immunofluorescence 1:50 - 1:200
  • Immunohistochemistry 1:250
  • Immunohistochemistry-Frozen 1:250
  • Immunohistochemistry-Paraffin 1:250-1:500
  • In vivo assay
  • Knockout Validated
  • Simple Western
  • Western Blot 1 - 2 ug/ml
• 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). Simple Western reported by an internal validation. Separated by Size
• 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:

  Mouse Eye Tissue Lysate (Normal)

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) - BSA Free

• 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.

Publications for RPE65 Antibody (NB100-355)(87)

Publications using NB100-355

• Jo D, Jang H, Cho C, et al. Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis bioRxiv 2021-01-10 (ICC/IF, Mouse)
• Wu J, Cho CS, Jo DH, Kim JH Application of Base Editor-Mediated Genome Editing in Mouse Retina Methods in molecular biology (Clifton, N.J.) 2023-01-02 [PMID: 36592316]
• Jo DH, Jang HK, Cho CS et al. Visual function restoration in a mouse model of Leber congenital amaurosis via therapeutic base editing Molecular therapy. Nucleic acids 2023-03-14 [PMID: 36589710] (IHC-P, Mouse)
  
  Details:
  Dilution used in IHC-P 1:100
• 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 2019-01-01 (IF/IHC, Human)
  
  Details:
  Citation using the Alexa Fluor 488 version of this antibody.
• Ripolles-Garcia A, Chen Y, Sato Y et al. Retinal Vascular Plexuses Are Unequally Affected in Canine Inherited Retinal Degenerations Investigative ophthalmology & visual science 2022-11-01 [PMID: 36378130] (IHC-Fr, Canine)
  
  Details:
  Dilution used in IHC-Fr 1:500
• Tian H, Chen Z, Zhu X et al. Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration iScience 2022-09-01 [PMID: 36185374] (WB, IHC-Fr, Human)
  
  Details:
  WB dilution used 1:1000
• SimOn MV, Vera MS, Tenconi PE et al. Sphingosine-1-phosphate and ceramide-1-phosphate promote migration, pro-inflammatory and pro-fibrotic responses in retinal pigment epithelium cells Experimental eye research 2022-08-27 [PMID: 36041511] (ICC/IF, Human)
• Nguyen Hoang AT, Lee H, Lee SJ Casein kinase I inhibitor D4476 influences autophagy and apoptosis in chloroquine-induced adult retinal pigment epithelial-19 cells Experimental eye research 2022-02-24 [PMID: 35219693] (IF/IHC, Mouse)
• Nizawa T, Bhutto IA, Tiwari A et al. Topical Ketotifen Fumarate Inhibits Choroidal Mast Cell Degranulation and Loss of Retinal Pigment Epithelial Cells in Rat Model for Geographic Atrophy Translational vision science & technology 2021-12-01 [PMID: 34967831]
• Zhang J, Gao F, Ma Y Et al. Identification of early-onset photoreceptor degeneration in transgenic mouse models of Alzheimer's disease iScience 2021-11-01 [PMID: 34805789] (IF/IHC, Mouse)
• Chae JB, Jang H, Son C Et al. Targeting senescent retinal pigment epithelial cells facilitates retinal regeneration in mouse models of age-related macular degeneration GeroScience 2021-10-02 [PMID: 34601706] (IF/IHC, Mouse)
• Jang HK, Jo DH, Lee SN Et al. High-purity production and precise editing of DNA base editing ribonucleoproteins Science advances 2021-08-01 [PMID: 34452911] (ICC/IF, Mouse)
• Zhao Q, Kong Y, Kittredge A et al. Distinct expression requirements and rescue strategies for BEST1 loss- and gain-of-function mutations eLife 2021-06-01 [PMID: 34061021] (WB)
• Kambhampati SP, Bhutto IA, Wu T et al. Systemic dendrimer nanotherapies for targeted suppression of choroidal inflammation and neovascularization in age-related macular degeneration Journal of controlled release : official journal of the Controlled Release Society 2021-05-28 [PMID: 34058271]
• Tsai YT, Li Y, Ryu J et al. Impaired cholesterol efflux in retinal pigment epithelium of individuals with juvenile macular degeneration American journal of human genetics 2021-04-21 [PMID: 33909993]
• You J, Cheng Y, Yang XJ, Chen L Generation of a homozygous LRP2 knockout human embryonic stem cell line (FDCHDPe010-A-56) by CRISPR/Cas9 system Stem cell research 2021-04-08 [PMID: 33878707]
• Chen Y, Huang Y, Wu P et al. The Functional Vision Protection Effect of Danshensu via Dopamine D1 Receptors: In Vivo Study Nutrients 2021-03-17 [PMID: 33803057] (IHC-P, Mouse)
• Chen J, Sun J, Hu Y et al. MicroRNA-191-5p ameliorates amyloid-beta 1-40 -mediated retinal pigment epithelium cell injury by suppressing the NLRP3 inflammasome pathway FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2021-04-01 [PMID: 33715208] (ICC/IF, Mouse)
• 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 2020-12-08 [PMID: 33301813] (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 2020-07-01 [PMID: 32791386] (IF/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 2020-04-25 [PMID: 32344885] (ICC/IF, 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. 2020-06-01 [PMID: 32196081] (IHC-Fr, Mouse)
• Yang C, Shani S, Tahiri H et Al. Extracellular microparticles exacerbate oxidative damage to retinal pigment epithelial cells Exp. Cell Res. 2020-03-13 [PMID: 32173468] (IF/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. 2020-01-21 [PMID: 31964819] (WB, Mouse)
• Ji C, Li Y, Kittredge A, et al. Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations Sci Rep 2019-12-13 [PMID: 31836750] (WB, Human)
• Smith JR, Ashander LM, Ma Y et al. Model Systems for Studying Mechanisms of Ocular Toxoplasmosis Methods Mol. Biol. 2019-11-24 [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. 2019-10-05 [PMID: 31673221] (ICC/IF, Human)
• Weed L, Ammar MJ, Zhou S et al. Safety of Same-Eye Subretinal Sequential Readministration of AAV2-hRPE65v2 in Non-human Primates Mol Ther Methods Clin Dev 2019-12-13 [PMID: 31660416] (IHC-Fr, Primate)
  
  Details:
  The primate species used as part of this study included two cynomolgus and one macaque.
• Gardiner KL, Cideciyan AV, Swider M et al. Long-Term Structural Outcomes of Late-Stage RPE65 Gene Therapy Mol. Ther. 2019-09-03 [PMID: 31604676] (IF/IHC, Canine)
• Lian C, Lou H, Zhang J et al. MicroRNA-24 protects retina from degeneration in rats by down-regulating chitinase-3-like protein 1 Exp. Eye Res. 2019-09-03 [PMID: 31491426] (WB, Rat, Human)
• Hellinen L, Pirskanen L, Tengvall-Unadike U et al. Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties Pharmaceutics 2019-08-13 [PMID: 31412689] (ICC/IF)
• Wright T, Brisson BA, Wood GA et al Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs. Cytometry A. 2019-09-01 [PMID: 31282052]
  
  Details:
  Citation using the FITC version of this antibody.
• 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 2019-08-01 [PMID: 31004408] (IF/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. 2019-04-10 [PMID: 30980814] (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 2018-09-22 [PMID: 30244402] (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. 2018-03-20 [PMID: 30208410] (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. 2018-08-20 [PMID: 30126999] (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 2018-08-01 [PMID: 30091312] (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. 2018-07-26 [PMID: 30049796]
• Marmorstein A. D, Johnson A. A, et al. Mutant Best1 Expression and Impaired Phagocytosis in an iPSC Model of Autosomal Recessive Bestrophinopathy. Sci Rep 2018-03-14 [PMID: 29540715] (SW, Human)
• Llonch S, Carido M, Ader M. Organoid technology for retinal repair. Dev. Biol. 2018-01-15 [PMID: 29291970] (IF/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. 2017-10-24 [PMID: 29063836] (WB, Human)
• Liao R, Yan F, Zeng Z et al. IGF-1 activates PI3K/Akt signaling to protect human retinal pigment epithelial cells from amiodarone-induced oxidative injury Br. J. Pharmacol. 2017-10-22 [PMID: 29057462] (IF/IHC, Rat)
• Sun J, Huang P, Liang J et al. Cooperation of Rel family members in regulating Ab1-40-mediated pro-inflammatory cytokine secretion by retinal pigment epithelial cells Cell Death Dis. 2017-10-12 [PMID: 29022897] (ICC/IF, Mouse)
• Steinfeld J, Steinfeld I, Bausch A et al. BMP-induced reprograming of the retina into RPE requires WNT signalling in the developing chick optic cup. Biol Open 2017-05-25 [PMID: 28546339] (IF/IHC, Chicken)
• Chowers G, Cohen M, Marks-Ohana D et al. Course of Sodium Iodate-Induced Retinal Degeneration in Albino and Pigmented Mice. Invest. Ophthalmol. Vis. Sci. 2017-04-01 [PMID: 28418497] (IF/IHC, Mouse)
• Zheng W, Meng Q, Wang H et al. IGF-1-Mediated Survival from Induced Death of Human Primary Cultured Retinal Pigment Epithelial Cells Is Mediated by an Akt-Dependent Signaling Pathway. Mol. Neurobiol. 2017-02-25 [PMID: 28238097] (Human)
• Sudharsan R, Simone K M et al. Acute and Protracted Cell Death in Light-Induced Retinal Degeneration in the Canine Model of Rhodopsin Autosomal Dominant Retinitis Pigmentosa. Invest Ophthalmol Vis Sci 2017-01-01 [PMID: 28114588] (IF/IHC, Canine)
• Zhou TE, Rivera JC, Bhosle VK et al. Choroidal Involution Is Associated with a Progressive Degeneration of the Outer Retinal Function in a Model of Retinopathy of Prematurity: Early Role for IL-1B. Am. J. Pathol. 2016-10-18 [PMID: 27768863] (IF/IHC)
• Lyzogubov VV, Bora PS, Wu X et al. The Complement Regulatory Protein CD46 Deficient Mouse Spontaneously Develops Dry-Type Age-Related Macular Degeneration-Like Phenotype. Am. J. Respir. Cell Mol. Biol. 2016-06-11 [PMID: 27295359] (ICC/IF)
  
  Details:
  This citation used the Alexa Fluor 647 version of this antibody.
• Iwabe S, Ying GS, Aguirre GD, Beltran WA. Assessment of visual function and retinal structure following acute light exposure in the light sensitive T4R rhodopsin mutant dog. Exp. Eye Res. 2016-04-13 [PMID: 27085210] (IHC-Fr, Canine)
• Di Lauro S, Rodriguez-Crespo D, Gayoso MJ et al. A novel coculture model of porcine central neuroretina explants and retinal pigment epithelium cells. Mol Vis 2016-03-25 [PMID: 27081295]
• Ayala-Pena VB, Pilotti F, Volonte Y et al. Protective effects of retinoid x receptors on retina pigment epithelium cells Biochim. Biophys. Acta 2016-02-13 [PMID: 26883505] (ICC/IF, Human)
• Chen M, Rajapakse D, Fraczek M et al. Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis Aging Cell 2016-02-15 [PMID: 26875723] (ICC/IF, Mouse)
• Parker J, Mitrousis N, Shoichet MS. Hydrogel for Simultaneous Tunable Growth Factor Delivery and Enhanced Viability of Encapsulated Cells in Vitro. Biomacromolecules. 2016-02-08 [PMID: 26762290] (ICC/IF, Human)
• Cashman SM, Gracias J, Adhi M, Kumar-Singh R. Adenovirus mediated delivery of factor H attenuates complement C3 induced pathology in the murine retina: a potential gene therapy for AMD. J Gene Med 2015-09-15 [PMID: 26369397] (IHC-P, Mouse)
• Lee S H, Kim H D et al. Time-Dependent Changes of Cell Proliferation After Laser Photocoagulation in Mouse Chorioretinal Tissue. Invest Ophthalmol Vis Sci 2015-01-04 [PMID: 25813995] (IF/IHC, Mouse)
• Yan RT, He L, Zhan W, Wang SZ. Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin PLoS ONE. 2015-01-31 [PMID: 25635399] (IHC-Fr, Mouse)
• Prasad T, Verma A, Li Q. Expression and cellular localization of the Mas receptor in the adult and developing mouse retina. Mol. Vis. 2014-10-29 [PMID: 25352750] (IHC-P, Mouse)
• Steinfeld J, Steinfeld I, Coronato N et al. RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling. Development. 2013-12-01 [PMID: 24227655] (IHC-Fr, ICC/IF, Chicken)
• Guziewicz KE, Zangerl B, Komaromy AM et al. Recombinant AAV-mediated BEST1 transfer to the retinal pigment epithelium: analysis of serotype-dependent retinal effects PLoS One. 2013-10-15 [PMID: 24143172] (IHC-Fr, Canine)
• Inoue Y, Hasegawa S, Yamada T et al. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway. PLoS One 2013-06-21 [PMID: 23805217] (ICC/IF, Mouse)
• Singh AK, Srivastava GK, Martin L et al. Bioactive substrates for Human retinal pigment epithelial cell growth from elastin-like recombinamers. J Biomed Mater Res A 2013-03-29 [PMID: 23554132] (ICC/IF, Human)
• Shirasawa M, Sonoda S, Terasaki H et al. TNF-alpha disrupts morphologic and functional barrier properties of polarized retinal pigment epithelium. Exp Eye Res 2013-02-27 [PMID: 23454586] (WB, Porcine)
• Wallace CA, Moir G, Malone D et al. Regulation of T-lymphocyte CCL3 and CCL4 production by retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2013-01-08 [PMID: 23299481]
• Fernandez-Bueno I, Rodriguez de la Rua E, Hileeto D. Histology and immunochemistry evaluation of autologous translocation of retinal pigment epithelium-choroid graft in porcine eyes Acta Ophthalmol 2012-12-19 [PMID: 23256869] (IF/IHC, ICC/IF, Porcine)
• He Y, Ge J, Burke JM et al. Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress. J Ocul Biol Dis Infor 2010-09-01 [PMID: 22833778] (ICC/IF, Human)
• Jiang G, Sun D, Kaplan HJ, Shao H. Retinal Astrocytes Pretreated with NOD2 and TLR2 Ligands Activate Uveitogenic T Cells plos One 2012-01-01 [PMID: 22808176] (ICC/IF, Mouse)
• Cho MS, Kim SJ, Ku SY et al. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses Stem Cell Res 2012-05-16 [PMID: 22683799] (ICC/IF, Human)
• Ukrohne TU, Westenskow PD, Kurihara T et al. Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells. Stem Cells Transl Med 2012-02-01 [PMID: 22532929]
• Xia H, Krebs MP, Kaushal S, Scott EW. Enhanced retinal pigment epithelium regeneration after injury in MRL/MpJ mice. Exp Eye Res;93(6):862-72. 2011-12-01 [PMID: 21989111] (IHC-Fr, Mouse)
• Kleinman ME, Kaneko H, Cho WG et al. Short-interfering RNAs induce retinal degeneration via TLR3 and IRF3. Mol Ther. 2012-01-01 [PMID: 21988875] (IF/IHC, Mouse)
  
  Details:
  Antibodies cited: 1. TLR3 pAb (IMG-516): IHC (P), Fig 4D (mouse eye sections) 2. TLR3-PE mAb (IMG-315D): Flow (Surface) and Flow (Intracellular), Fig 4F [primary human retinal pigment epithelium cell isolates (hRPE)]. TLR3 was expressed on both the cell s
• Leung KW, Gvritishvili A, Liu Y, Tombran-Tink J. ZIP2 and ZIP4 mediate age-related zinc fluxes across the retinal pigment epithelium. J Mol Neurosci;46(1):122-37. 2012-01-01 [PMID: 21603979] (FLOW, IF/IHC, ICC/IF, Mouse, Human)
• Harness JV, Turovets NA, Seiler MJ et al. Equivalence of conventionally-derived and parthenote-derived human embryonic stem cells. PLoS ONE;6(1). 2011-01-01 [PMID: 21249129] (ICC/IF, Human)
• Hernandez M, Pearce-Kelling SE, Rodriguez FD et al. Altered expression of retinal molecular markers in the canine RPE65 model of Leber congenital amaurosis. Invest Ophthalmol Vis Sci 2010-12-01 [PMID: 20671290]
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Reviews for RPE65 Antibody (NB100-355) (7)

Average Rating: 3.4

(Based on 7 reviews)

We have 7 reviews tested in 3 species: Human, Bovine and Human, Other.

reviewed by: Theresa Smith IP Bovine and Human 05/06/2019

Summary

• Application: Immunoprecipitation
• Sample Tested: 293T cell lysate (overexpressing RPE65),Bovine RPE cell extracts
• Species: Bovine and Human
• Lot: N

Comments

• Comments: We 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

reviewed by: Verified Customer ICC Human 08/29/2017

Summary

• Application: Immunocytochemistry
• Sample Tested: ARPE-19 cells
• Species: Human
• Lot: J-1

Comments

• Comments: Fixative Methanol
  Blocking Goat Serum
  Dilution 1:250
  Incubation 1h RT

reviewed by: Abbie Jensen IF Other 03/23/2016

Summary

• Application: Immunofluorescence
• Sample Tested: pfa fixed 11day old zebrafish cryosections
• Species: Other

reviewed by: cynthia morimoto IHC-Fr Other 12/12/2014

Summary

• Application: Immunohistochemistry-Frozen
• Sample Tested: See PMID 24143172
• Species: Other

reviewed by: Verified Customer IF Other 10/06/2014

Summary

• Application: Immunofluorescence
• Species: Other

reviewed by: Verified Customer IF Human 04/30/2014

Summary

• Application: Immunofluorescence
• Species: Human

reviewed by: Sujoy Bhattacharya WB Human 12/06/2010

Summary

• Application: Western Blot
• Sample Tested: RPE whole cell lysate, Sample Amount: 20-25microgram
• Species: Human

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.

Bioinformatics

• Gene Symbol: RPE65