title: An easy, rapid method to isolate RPE cell protein from the mouse eye.
abstract: The retinal pigment epithelium (RPE) is essential for maintaining the health of the neural retina. RPE cell dysfunction plays a critical role in many common blinding diseases including age-related macular degeneration (AMD), diabetic retinopathy, retinal dystrophies. Mouse models of ocular disease are commonly used to study these blinding diseases. Since isolating the RPE from the choroid has been challenging, most techniques separate the RPE from the retina, but not the choroid. As a result, the protein signature actually represents a heterogeneous population of cells that may not accurately represent the RPE response. Herein, we describe a method for separating proteins from the RPE that is free from retinal and choroidal contamination. After removing the anterior segment and retina from enucleated mouse eyes, protein from the RPE was extracted separately from the choroid by incubating the posterior eyecup with a protein lysis buffer for 10 min. Western blot analysis identified RPE65, an RPE specific protein in the RPE lysates, but not in choroidal lysates. The RPE lysates were devoid of rhodopsin and collagen VI, which are abundant in the retina and choroid, respectively. This technique will be very helpful for measuring the protein signal from the RPE without retinal or choroidal contamination.
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Wei
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Zixian
authors.initials: Z
authors.lastname: Xun
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Herta
authors.initials: H
authors.lastname: Granado
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Wu
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. Electronic address: jthanda@jhmi.edu.
authors.forename: James T
authors.initials: JT
authors.lastname: Handa
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D004210:Dissection / Q000379:methods*; D005136:Eye Proteins / Q000302:isolation & purification*; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D055213:Retinal Pigment Epithelium / Q000737:chemistry* / Q000601:surgery
chemical_list: D005136:Eye Proteins
keywords: Choroid; Dissection; Mouse; Retinal pigmented epithelium