TY - JOUR
T1 - An easy, rapid method to isolate RPE cell protein from the mouse eye
AU - Wei, Hong
AU - Xun, Zixian
AU - Granado, Herta
AU - Wu, Angela
AU - Handa, James T.
N1 - Funding Information:
Funding: NIH EY14005 (JTH), EY019044 (JTH), RPB Senior Scientist Award (JTH), unrestricted award from RPB to the Wilmer Eye Institute; P30EY001765 core grant, and a gift from the Merlau family and Aleda Wright. JTH is the Robert Bond Welch Professor.
Publisher Copyright:
© 2015 Elsevier Ltd.
PY - 2016/4/1
Y1 - 2016/4/1
N2 - 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.
AB - 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.
KW - Choroid
KW - Dissection
KW - Mouse
KW - Retinal pigmented epithelium
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U2 - 10.1016/j.exer.2015.09.015
DO - 10.1016/j.exer.2015.09.015
M3 - Short survey
C2 - 26424220
AN - SCOPUS:84945971453
VL - 145
SP - 450
EP - 455
JO - Experimental Eye Research
JF - Experimental Eye Research
SN - 0014-4835
ER -