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2 protocols using non fat dried milk

1

Immunoblotting of Human Retina Proteins

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The immunoblots were performed as previously described (84 (link)). Briefly, one-fourth of a human retinae was placed in 125 μL HGNT-buffer, sonicated three times for 5 s and mixed with SDS-PAGE sample buffer (62.5 Mm Tris–HCL, pH 6.8; 10% glycerol, 2% SDS, 5% mercaptoethanol, 1 Mm EDTA and 0.025% bromphenol blue). As a molecular marker, PageRuler™ Prestained Protein Ladder ranging from 11 to 170 kDa was used (Fermentas). A total of 30 μg retina protein extracts were separated on 8% polyacrylamide gels and blotted onto PVDF transfer membranes (Millipore) followed by blocking with non-fat dried milk (Applichem). Cell pellets of enriched cell populations from retinal punches (6 Mm) were dissolved in reducing the Laemmli sample buffer, and denatured and sonicated. Samples were separated using a 12% SDS-PAGE. Detection was performed either by a chemiluminescence detection system (WesternSure PREMIUM Chemiluminescent Substrate, LI-COR) or using the Odyssey infra-red imaging system (LI-COR Biosciences).
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2

Western Blot Protein Analysis Protocol

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Protein extracts were prepared using a RIPA buffer (50 mM Tris/HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate (all from Sigma-Aldrich), 150 mM NaCl (Carl Roth, Karlsruhe, Germany), and 1% Triton X-100 (Roche, Penzberg, Germany)) with 5% protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted to equal concentrations with 1× Roti®-Load 1 (Carl Roth). SDS-PAGE was performed following standard protocols. The Pierce™ Power Blotter (Thermo Fisher Scientific) was used for semi-dry protein transfer to PVDF membranes (Pall, Port Washington, NY, USA). Membranes were blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany)/TBS-T (40 mM Tris/HCl (pH 7.6), 273 mM NaCl, 0.1% Tween® 20 (Sigma-Aldrich)) for 1 h at room temperature. Incubation with primary antibodies was performed in blocking solution overnight at 4 °C, and incubation with secondary antibodies was performed in TBS-T for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific), and signals were detected on a ChemiDoc Touch Imaging System (Bio-Rad). Bands were quantified using Image Lab v6 (Bio-Rad). The antibodies and the concentrations used are listed in Supplementary Table S2.
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