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

Novus Biologicals, part of Bio-Techne | Catalog # NB100-355

Novus Biologicals, part of Bio-Techne

Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat, Porcine, Bovine, Canine, Chicken, Primate, Xenopus

Cited:

Human, Mouse, Rat, Porcine, Avian - Chicken, Canine, Primate

Applications

Validated:

CyTOF-ready, Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Immunoprecipitation, In vivo assay, Knockout Validated, Simple Western, Western Blot

Cited:

Flow Cytometry, IF/IHC, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 kappa Clone # 401.8B11.3D9

Format

BSA Free

Concentration

1.0 mg/ml

View all RPE65 Antibodies »

Product Specifications

Immunogen

This RPE65 Antibody (401.8B11.3D9) was developed against bovine RPE65 microsomal membrane proteins. [UniProt# Q28175]

Reactivity Notes

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

Localization

Cytoplasmic, Microsomeal and Cell Membrane

Marker

Retinal Pigment Epithelium Marker

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1 kappa

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.

Scientific Data Images for RPE65 Antibody (401.8B11.3D9) - BSA Free

Western Blot: RPE65 Antibody (401.8B11.3D9)BSA Free [NB100-355]

Western Blot: RPE65 Antibody (401.8B11.3D9)BSA Free [NB100-355]

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 (https://elifesciences.org/articles/29914), licensed under a CC-BY license.
Immunocytochemistry/ Immunofluorescence: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355]

Immunocytochemistry/ Immunofluorescence: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355]

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]

Immunohistochemistry-Paraffin: RPE65 Antibody (401.8B11.3D9) - BSA Free [NB100-355]

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).
View 10 more images

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

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:50 - 1:200

Immunofluorescence

1:50 - 1:200

Immunohistochemistry

1:250

Immunohistochemistry-Frozen

1:250

Immunohistochemistry-Paraffin

1:250-1:500

Immunoprecipitation

reported by customer review

In vivo assay

reported in scientific literature (PMID 32173468)

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. See Simple Western Antibody Database for Simple Western validation: tested in iPSC-RPE; separated by size
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 7 reviews rated 3.4 using NB100-355 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: RPE65

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

Alternate Names

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

Gene Symbol

RPE65

Additional RPE65 Products

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

Product Datasheets

Certificate of Analysis

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Product Specific Notices for RPE65 Antibody (401.8B11.3D9) - BSA Free

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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