Retinal pigmented epithelium derived neurotrophic factor
a pigmented epithelium and neurotrophic factor technology, applied in the field of retina pigmented epithelium derived neurotrophic factor, can solve problems such as retinal degeneration
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example 1
Establishment of RPE Cell Cultures
[0151] RPE cells were harvested from post-mortem human eyes, as described by Pfeffer et al., J. Cell. Physiol. 117 333-341 (1983). The cells were grown in 75 cm flasks with Eagles's minimal essential medium supplemented with 15% v / v fetal calf serum and 5% v / v CO2 at 37° C. Cells were grown to confluence, harvested by trypsinizing the cell monolayer, and resuspended in MEM supplemented with 15% v / v fetal calf serum. A portion of the cells (about 5%) were reseeded into new 75 cm, where the cells were again grown in 5% v / v CO2 at 37° C.
example 2
Preparation of RPE-Conditioned Medium
[0152] Confluent cultures of RPE cells were washed extensively with HBSS, before conditioning, to remove serum proteins. Twenty-five ml of serum-free MEM was added, and the cultures were incubated with 5% v / v CO2 at 37° C. for about 48 hours. The conditioned medium was then collected and centrifuged at 1,000 rpm at room temperature, to remove any free cells and other particulate matter, to provide an impure PEDF protein fraction.
example 3
Purification of PEDF by SDS Gel Electrophoresis
[0153] Proteins contained in human fetal RPE-conditioned medium, prepared as described in Example 2, and in control non-conditioned medium were analyzed on an SDS-polyacrylamide slab gel.
[0154] The conditioned and non-conditioned media samples were mixed with an electrophoresis sample buffer (62.5 mM Tris, pH 6.8, 2% w / v SDS, 10% v / v glycerol, 0.001% w / v bromophenol blue, and 0.1 M 2-mercaptoethanol). Molecular-weight markers, such as phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme (such as that supplied by Bio-Rad), were also mixed with an electrophoresis sample buffer. All the samples were denatured at 100° C., in a boiling water bath, for 5 minutes and loaded onto a SDS containing 7.5% w / v polyacrylamide gel. The electrophoresis was conducted at 20 mA until the bromphenol blue marker dye migrated to the bottom of the gel.
[0155] At the completion of electrophoresis, strip...
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