Bestrophin 1 Antibody (E6-6) - BSA Free

Western Blot: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - Western blot analysis of the normal and mutant human Best1 protein in transiently transfected MDCK cells. Best1 proteins are detectable as a 68 kDa band in all transfected cells, but not in non-transfected controls (MDCK lane). Actin bands are shown to indicate equal loading of cell lysates. Image collected and cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/14/7/15121), licensed under a CC-BY license.

Immunocytochemistry/Immunofluorescence: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - X-Z confocal single image scan of transiently transfected cells with different BEST1 cDNA constructs showing mislocalization of mutants Y85H, Q96R, L100R and Y227N. Cells were stained for Best1 (green), beta-catenin (red) and nuclei (blue). Scale bar = 10 um. Image collected and cropped by CiteAb from the following publication (//www.mdpi.com/1422-0067/14/7/15121), licensed under a CC-BY license.

Immunohistochemistry-Paraffin: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - Bestrophin 1 was detected in immersion fixed paraffin sections of human small intestine using t Mouse Anti-Human Bestrophin 1 Monoclonal Antibody (Catalog # NB300-164) at 5 ug/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cell surface and extracellular.

Western Blot: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - Detection of Bestrophin (68 kDa) from human RPE cell lysate.

Immunohistochemistry-Paraffin: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - Bestrophin 1 was detected in immersion fixed paraffin-embedded sections of human brain using Mouse Anti-Human Bestrophin 1 (E6-6) Monoclonal Antibody (Catalog # NB300-164) at 1:300 for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to the cytoplasm in neurons.

Knockout Validated: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - 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 license.

Dual RNAscope ISH-IHC: Bestrophin 1 Antibody (E6-6) - BSA Free [NB300-164] - Formalin-fixed paraffin-embedded tissue sections of human duodenum were probed for Bestrophin 1 mRNA (ACD RNAScope Probe, catalog # 433181; Fast Red chromogen, ACD catalog # 322360). Adjacent tissue section was processed for immunohistochemistry using mouse anti-human (Novus Biologicals catalog # NB300-164) at 0.3ug/mL with overnight incubation at 4 degrees Celsius followed by incubation with anti-mouse IgG VisUCyte HRP Polymer Antibody (Catalog # VC001) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). IHC signal is confined to cytoplasm.

• Reactivity: Hu, Mu, Po, Ca, Pm, Rt(-)
• Applications: WB, Func, ICC/IF, IHC, IP, Dual ISH-IHC, KO
• Clone: E6-6
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

Bestrophin 1 Antibody (E6-6) - BSA Free Summary

• Immunogen: Synthetic peptide conjugated to KLH corresponding to the C-terminus of human Bestrophin 1 (KDHMDPYWALENRDEAHS) [Uniprot: O76090]
• Isotype: IgG1 Kappa
• Clonality: Monoclonal
• Host: Mouse
• Gene: BEST1
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • Dual RNAscope ISH-IHC
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry reported in scientific literature (PMID 30048622)
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin reported in scientific literature (PMID 24345323)
  • Immunoprecipitation
  • Knockout Validated
  • Proximity Ligation Assay reported in scientific literature (PMID 27519691)
  • Western Blot 1:1000
• Application Notes: In Western blot, this antibody recognizes a band at ~68 kDa representing Bestrophin. Please see protocol for treatment of cell extracts. 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.

Reactivity Notes

Human, Primate, Porcine reactivity reported in scientific literature (PMID: 11050159). Use in Mouse reported in scientific literature (PMID:32791386).

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 A or G purified

Alternate Names for Bestrophin 1 Antibody (E6-6) - BSA Free

• ARB
• Best disease
• BEST
• bestrophin 1
• bestrophin-1
• BMD
• TU15B
• vitelliform macular dystrophy 2
• Vitelliform macular dystrophy protein 2
• VMD2RP50

Background

Best macular dystrophy (BMD) or vitelliform macular dystrophy (VMD2), is an autosomal form of macular degeneration, characterized by a depressed light peak in the electrooculogram (EOG). It is inherited and has an early onset. Bestrophin is a 68 kDa basolateral plasma membrane protein encoded by the VMD2 gene. Bestrophin's function is still unknown, but data suggests that it is a chloride channel that plays a role in generating the altered EOG in Best disease patients. In addition, Bestrophin is a useful biochemical and histological marker of RPE (retinal pigment epithelial cells) cells.

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 Bestrophin 1 Antibody (NB300-164)(47)

Publications using NB300-164

• Li J, Qiu C, Wei Y et al. Human Amniotic Epithelial Stem Cell-Derived Retinal Pigment Epithelium Cells Repair Retinal Degeneration Frontiers in Cell and Developmental Biology 2021-09-28 [PMID: 34650985]
• Sripathi SR, Hu MW, Turaga RC Et al. Epithelial to mesenchymal transition (EMT) of human stem cell-derived retinal pigment epithelium shares commonalities with malignancy-associated EMT: a proteomic analysis Molecular & cellular proteomics : MCP 2021-08-26 [PMID: 34455105] (WB, Human)
• Afanasyeva TAV, Corral-Serrano JC, Garanto A et al. A look into retinal organoids: methods, analytical techniques, and applications Cellular and Molecular Life Sciences 2021-10-01 [PMID: 34420069]
• Schustak J, Twarog M, Wu X et al. Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE Journal of Immunology Research 2021-06-18 [PMID: 34239945] (ICC/IF, Human)
• Choudhury R, Bayatti N, Scharff R Et Al. FHL-1 interacts with human RPE cells through the alpha 5 beta 1 integrin and confers protection against oxidative stress Scientific reports 2021-07-08 [PMID: 34239032] (ICC/IF)
• 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)
• 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]
• D'Antonio-Chronowska, A, D'Antonio, M Et al. In vitro Differentiation of Human iPSC-derived Retinal Pigment Epithelium Cells (iPSC-RPE). Bio Protoc 2019-12-20 [PMID: 33654959] (WB, Mouse)
• Biswas P, Borooah S, Matsui H et al. Detection and validation of novel mutations in MERTK in a simplex case of retinal degeneration using WGS and hiPSC-RPEs model Human mutation 2020-11-30 [PMID: 33252167]
• Bonilha, V L, Bell, B A Et al. Cellular Changes in Retinas From Patients With BEST1 Mutations. Front Cell Dev Biol 2020-11-07 [PMID: 33154968] (ICC/IF)
• SchAfer N, Wolf HN, Enzbrenner A et al. Properdin Modulates Complement Component Production in Stressed Human Primary Retinal Pigment Epithelium Cells Antioxidants (Basel) 2020-08-26 [PMID: 32859013] (IF/IHC, Human)
• 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] (ICC/IF, Mouse)
• Mladenov N, Petrova SD, Mladenova K et al. Miscibility of hBest1 and sphingomyelin in surface films - A prerequisite for interaction with membrane domains Colloids Surf B Biointerfaces 2020-02-21 [PMID: 32113084] (WB)
• Voisin A, Monville C, Plancheron A, et al. Cathepsin B pH-Dependent Activity Is Involved in Lysosomal Dysregulation in Atrophic Age-Related Macular Degeneration Oxid Med Cell Longev 2019-12-06 [PMID: 31885803] (Human)
• 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, IF/ICC, Human)
• Smith E, D'Antonio-Chronowska A, Greenwald W et al. Human iPSC-derived retinal pigment epithelium: a model system for identifying and functionally characterizing causal variants at AMD risk loci Stem Cell Reports 2019-05-14 [PMID: 31080113]
• Shahi PK, Hermans D, Sinha D et al. Gene Augmentation and Readthrough Rescue Channelopathy in an iPSC-RPE Model of Congenital Blindness Am. J. Hum. Genet. 2019-02-07 [PMID: 30686507] (ICC/IF, Human)
• Zhang Y, Kittredge A, Ward N et al. ATP activates bestrophin ion channels through direct interaction Nat Commun 2018-08-07 [PMID: 30087350] (ICC/IF, Human)
• Voisin A, Monville C, Plancheron A et al. HRPE cells derived from induced pluripotent stem cells are more sensitive to ozidative strss than ARPE-19 cell. Exp. Eye Res. 2018-07-23 [PMID: 30048622] (IF/IHC, Human)
• Ramachandra Rao S, Pfeffer BA, MAs GOmez N et al. Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome Autophagy. 2018-09-16 [PMID: 29979914] (WB, Human)
• 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] (Human)
• Zhang Y, Zhang D, Wei W et al. Effects of RPE-conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration. Exp Ther Med. 2017-10-01 [PMID: 29042966] (ICC/IF, Human)
• Klingeborn M, Dismuke WM, Skiba NP et al. Directional Exosome Proteomes Reflect Polarity-Specific Functions in Retinal Pigmented Epithelium Monolayers Sci Rep 2017-07-07 [PMID: 28687758] (ICC/IF, Porcine)
• Shim SH, Kim G, Lee DR et al. Survival of Transplanted Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Human Recipient for 22 Months. JAMA Ophthalmol. 2017-02-09 [PMID: 28196199]
• Kaneko Y, Pappas C, Tajiri N, Borlongan CV. Oxytocin modulates GABAAR subunits to confer neuroprotection in stroke in vitro. Sci Rep 2016-10-21 [PMID: 27767042]
• Uggenti C, Briant K, Streit AK et al. Restoration of mutant bestrophin-1 expression, localisation and function. Dis Model Mech. 2016-08-12 [PMID: 27519691] (WB, ICC/IF, Human, Canine)
• 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)
• Bertolotti E, Neri A, Camparini M et al. Stem cells as source for retinal pigment epithelium transplantation. Prog Retin Eye Res. 2014-06-13 [PMID: 24933042] (ICC/IF, Human)
  
  Details:
  Figure 4. Basolateral localization of bestrophin 1 (antibody used 1:1000, Novus Biologicals) in human RPE cells. Human retinal stem cells (RNS; derived from adult ciliary bodies of post-mortem donors) were differentiated into RPE and the molecular characteristics of RPE cells were evaluated - polarization of the RPE cells was assessed with three markers: bestrophin 1 at the basolateral membrane, and beta5-integrin as well as ezrin at the apical side of the RPE.
• Sharon D, Al-Hamdani S, Engelsberg K et al. Ocular phenotype analysis of a family with biallelic mutations in the BEST1 gene. Am J Ophthalmol 2013-12-14 [PMID: 24345323] (ICC/IF, IHC-P, Human)
• Doumanov J A, Zeitz C et al. Disease-causing mutations in BEST1 gene are associated with altered sorting of bestrophin-1 protein. Int J Mol Sci 2013-07-22 [PMID: 23880862] (ICC/IF, WB, Rat, Canine)
• Maruotti J, Wahlin K, Gorrell D et al. A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Transl Med 2013-05-01 [PMID: 23585288]
• Blenkinsop TA, Salero E, Stern JH, Temple S. The culture and maintenance of functional retinal pigment epithelial monolayers from adult human eye Methods Mol Biol 2013-01-01 [PMID: 23097100]
• 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] (ICC/IF, Human)
• Guziewicz KE, Slavik J, Lindauer SJ et al. Molecular consequences of BEST1 gene mutations in canine multifocal retinopathy predict functional implications for human bestrophinopathies. Invest Ophthalmol Vis Sci. 2011-04-15 [PMID: 21498618] (WB, Canine)
• Davidson AE, Millar ID, Burgess-Mullan R et al. Functional characterization of bestrophin-1 missense mutations associated with autosomal recessive bestrophinopathy. Invest Ophthalmol Vis Sci 2011-05-01 [PMID: 21330666]
• 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, WB, Human)
• Zhang Y, Patil RV, Marmorstein AD. Bestrophin 2 is expressed in human non-pigmented ciliary epithelium but not retinal pigment epithelium. Mol Vis;16:200-6. 2010-02-10 [PMID: 20157619] (WB, Human)
• Feng Q, Lu SJ, Klimanskaya I et al. Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cells 2010-04-01 [PMID: 20155819] (ICC/IF, Human)
• Davidson AE, Millar ID, Urquhart JE et al. Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa. Am J Hum Genet 2009-11-01 [PMID: 19853238] (Human)
• Buchholz DE, Hikita ST, RowlTJ et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells 2009-10-01 [PMID: 19658190] (WB, Human)
• Lu B, Malcuit C, Wang S et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 2009-09-01 [PMID: 19521979] (WB, Human)
• Guziewicz KE, Zangerl B, Lindauer SJ, Mullins RF, Sandmeyer LS, Grahn BH, Stone EM, AclGM, Aguirre GD. Bestrophin gene mutations cause canine multifocal retinopathy: a novel animal model for best disease. Invest Ophthalmol Vis Sci;48(5):1959-67. 2007-05-01 [PMID: 17460247] (WB, ICC/IF, Canine)
• Mullins RF, Oh KT, Heffron E et al. Late development of vitelliform lesions and flecks in a patient with best disease: clinicopathologic correlation. Arch Ophthalmol 2005-11-01 [PMID: 16286623]
• Klimanskaya, I, Hipp, J, Rezai, K et al. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells;6(3):217-45. 2004-01-01 [PMID: 15671670] (ICC/IF, Human)
• Marmorstein LY, McLaughlin PJ, Stanton JB, Yan L, Crabb JW, Marmorstein AD. Bestrophin interacts physically and functionally with protein phosphatase 2A. J Biol Chem;277(34):30591-7. 2002-08-23 [PMID: 12058047] (WB, Porcine)
• Marmorstein AD, Marmorstein LY, Rayborn M, Wang X, Hollyfield JG, Petrukhin K. Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium. Proc Natl Acad Sci U S A;97(23):12758-63. 2000-11-07 [PMID: 11050159] (IP, WB, IHC-Fr, ICC/IF, Primate, Porcine, Human)

Review for Bestrophin 1 Antibody (NB300-164) (1)

Average Rating: 5

(Based on 1 review)

We have 1 review tested in 1 species: Human and Rat.

reviewed by: Verified Customer WB Human and Rat 12/29/2016

Summary

• Application: Western Blot
• Sample Tested: RPE whole cell lysate
• Species: Human and Rat

Comments

• Comments: E6-6 used at 1:2000 over night. next day secondary antibody donkey anti-mouse peroxidase was applied followed by detection using enhanced chemiluminescence.

FAQs for Bestrophin 1 Antibody (NB300-164). (Showing 1 - 4 of 4 FAQs).

1. According to the data sheet, for Immunohistochemistry, it references (PMID: 21249129). I see a 1:25 dilution in the journal article referenced under immunocytochemistry. Is this the same dilution recommended for IHC? If not do you have a recommended dilution? Antigen retrieval: Do you have any information on AR? Citrate buffer or higher pH (EDTA)? Temperature? Time? Incubation: Is 1 hour at room temperature or overnight at 4C recommended?
   • This antibody was validated in IHC in the reference PMID 11050159. 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. I imagine the specific protocol can be found in the reference, however.
2. Can I get the recipe for RIPA buffer and the lysate preparation protocol for membrane bound proteins using NB300-164?
   • Here is both the recipe we use for RIPA and a good protocol for tissue lysate prep. RIPA buffer: 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. Preparation of lysate from tissues Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes and immerse in liquid nitrogen to snap freeze. Store samples at -80C for later use or keep on ice for immediate homogenization. For a approximately 5 mg piece of tissue, add approximately 300 ul lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2x300 ul lysis buffer, then maintain constant agitation for 2 hours at 4C (e.g place on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the amount of tissue present (protein extract should not be too diluted to avoid loss of protein and large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml). Centrifuge for 20 min at 12000 rpm at 4C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice; discard the pellet.
3. I want to know the concentration of Bestrophin 1 antibody [NB300-164].
   • For our product NB300-164, this antibody is provided as unpurified ascites. Therefore, the antibody concentration has not been determined.
4. We are interested in your item, NB300-164 and would you please let us know the antibody concentration of it?
   • The total protein concentration of the ascites (Lot# B-3) is 11.7 mg/ml.

Bioinformatics

• Gene Symbol: BEST1
• Entrez:
  • 7439(Human)
  • 293735(Rat)
• Uniprot:
  • O76090(Human)