Samples were collected in sample buffer containing 312.5 mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 12.5% 2-mercaptoethanol, bromophenol blue, and protease inhibitors (Sigma Cat#5056489001) and run on SDS-PAGE gels (8% PAGE gels for FLAG-Lphn immunoblots and 12% PAGE gels β-actin immunoblots) at 30 mA/gel constant current. The Precision Plus Protein Dual Color Protein Standard (BioRad Cat# 1610374) was used as a protein molecular weight ladder. Protein was transferred onto nitrocellulose transfer membrane in transfer buffer (25.1 mM Tris, 192 mM glycine, 20% methanol) at 200 mA constant current for 2 hr at 4°C. Membranes were blocked in 5% non-fat dry milk/TBST (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20) for 1 hr at room temperature, incubated in primary antibodies diluted into 5% milk/TBST overnight at 4°C, washed 3 × 5 min in TBST, incubated in corresponding secondary antibodies (Licor IRDye 800CW donkey anti-mouse Cat#92632212; IRDye 800CW Donkey anti-rabbit Cat#92632213) diluted into TBST, washed 5 × 5 min in TBST, and imaged on a Licor Odyssey system.
Precision plus protein dual color protein standard
Manufactured by Bio-Rad
The Precision Plus Protein Dual Color Protein Standard is a laboratory reagent used for protein molecular weight estimation. It contains a mixture of recombinant proteins with known molecular weights, which are pre-stained with two different dyes for easy visualization on protein gels.
Automatically generated - may contain errors
2 protocols using precision plus protein dual color protein standard
1
SDS-PAGE Immunoblotting Protocol
2
Western Blot Protein Detection Protocol
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Cells were briefly washed once with PBS, and samples were collected in sample buffer containing 312.5 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 12.5% 2-mercaptoethanol, bromophenol blue, and protease inhibitors (Sigma; Cat# 5056489001) and run on SDS-PAGE gels (12% PAGE gels for HA-PDE and β-actin IBs) at 30 mA/gel constant current. The Precision Plus Protein Dual Color Protein Standard (Bio-Rad; Cat# 1610374) was used as a protein molecular weight ladder. Protein was transferred onto nitrocellulose transfer membrane in transfer buffer (25.1 mM Tris, 192 mM glycine, and 20% methanol) at 200 mA constant current for 2 h at 4°C. Membranes were blocked in 5% nonfat dry milk/TBS with Tween-20 (TBST; 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween-20) for 1 h at room temperature, incubated in primary antibodies diluted into 5% milk/TBST overnight at 4°C, washed 3 × 5 min in TBST, incubated in corresponding secondary antibodies (Licor IRDye 800CW donkey anti-mouse; Cat# 92632212; IRDye 800CW donkey anti-rabbit Cat# 92632213) diluted into TBST, washed 5 × 5 min in TBST, and imaged on a Licor Odyssey system.
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