Western blot was performed on proteins extracted from a single retina [16 (link)]. Between seven and sixteen individuals were tested per condition. Harvested retinas were snap frozen before lysis in 80 μL P300 buffer (20 mM Na2HPO4; 250 mM NaCl; 30 mM NaPPi; 0.1% Nonidet P-40; 5 mM EDTA; 5 mM DTT) and protease inhibitor cocktail (Sigma-Aldrich, Saint-Quentin Fallavier, France). Protein content was measured using the Lowry protein assay kit (DC Protein Assay; Bio-Rad, Marnes-la-Coquette, France). Homogenates were sonicated and centrifuged for 15 min at 5 000 g, and then 10 μg of the supernatant were subjected to SDS-PAGE, as previously described [12 (link)]. Western blots were then conducted using standard procedures. Primary and secondary antibodies are listed in Supplementary Table S3 . Antibody binding was revealed by the Enhanced Chemiluminescence System (Bio-Rad) on X-Ray film (Sigma-Aldrich). Each sample was probed once with anti-α-tubulin antibody for normalization. Quantification was done using ImageJ software [71 (link)]. All original western blot gels are depicted in Supplementary Fig. 4 .
Lowry protein assay kit dc protein assay
Manufactured by Bio-Rad
Sourced in France
The Lowry protein assay kit (DC Protein Assay) is a colorimetric-based method for determining the total protein concentration in a sample. It measures the absorbance of the sample at a specific wavelength, which is proportional to the protein content. The kit provides the necessary reagents and protocols to perform the protein quantification assay.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (2 mentions)
X ray film, by Merck Group (1 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
3 protocols using lowry protein assay kit dc protein assay
1
Western Blot Analysis of Retinal Proteins
2
Western Blot Analysis of Retinal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed on protein extracts from single retinas, at least on three individuals per condition, unless otherwise specified in the figure legends. Retinas from enucleated eyes were dissected in Hanks’ Balanced Salt solution (Gibco) by removing the anterior segment, vitreous body, sclera and RPE and were frozen at −80 °C. Retinas were lysed in P300 buffer (20 mM Na2HPO4; 250 mM NaCl; 30 mM NaPPi; 0.1% Nonidet P-40; 5 mM EDTA; 5 mM DTT) supplemented with protease inhibitor cocktail (Sigma–Aldrich). For RPE protein extracts, the anterior segment and the retina were removed from enucleated mouse eyes. The RPE was then separated from the choroid by incubating the posterior eyecup (sclera-choroid-RPE) with P300 buffer for 10 min. Protein concentration was determined using a Lowry protein assay kit (DC Protein Assay; Bio-Rad). Equal amounts of proteins (20 µg/lane) of each sample were loaded, separated by 7.5% SDS-PAGE (Bio-Rad) and transferred onto nitrocellulose membranes. Western blots were then conducted using standard procedures. Primary and secondary antibodies are listed in Supplementary Table S2 . An enhanced chemiluminescence kit (Bio-Rad) was used to detect the proteins. Each sample was probed once with anti-α-tubulin antibody for normalisation. Quantification was done using Fiji software (National Institutes of Health24 (link)).
3
Western Blot Analysis of Retinal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed on protein extracts from single retinas, at least on 3 individuals per condition, unless otherwise specified in the figure legends. Retinas from enucleated eyes were dissected in Hanks' Balanced Salt solution (Gibco) by removing the anterior segment, vitreous body, sclera and RPE and were frozen at -80°C. Retinas were lysed in P300 buffer (20 mM Na 2 HPO 4 ; 250 mM NaCl; 30 mM NaPPi; 0.1% Nonidet P-40; 5 mM EDTA; 5mM DTT) supplemented with protease inhibitor cocktail (Sigma-Aldrich). For RPE protein extracts, the anterior segment and the retina were removed from enucleated mouse eyes. The RPE was then separated from the choroid by incubating the posterior eyecup (sclera-choroid-RPE) with P300 buffer for 10 min. Protein concentration was determined using a Lowry protein assay kit (DC Protein Assay; Bio-Rad). Equal amounts of proteins (20 µg/lane) of each sample were loaded, separated by 7.5% SDS-PAGE (Bio-Rad) and transferred onto nitrocellulose membranes. Western blots were then conducted using standard procedures. Primary and secondary antibodies are listed in Supplementary Table S2. An enhanced chemiluminescence kit (Bio-Rad) was used to detect the proteins. Each sample was probed once with anti-a-tubulin antibody for normalization. Quantification was done using Fiji software (National Institutes of Health, (Schindelin et al., 2012) ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!