Membrane proteins were transferred from BN-PAGE to PVDF membranes. After blocking of the membranes for 1 h with 10% non-fat dry milk in 0.1% TBST (100 mM Tris–HCL, 150 mM NaCl, pH 7.5, 0.1% Tween 20), mouse anti-GABAB1 (Abcam, ab55051, Cambridge, UK; 1/5000). Goat anti-GABAB1a (sc-73401/2500), goat anti-MuscarinicM2 (Abcam, ab140473, Cambridge, UK; 1/3000) and rabbit anti-GABAB2 (Abcam, ab75838, Cambridge, UK; 1/5000). The antibodies were detected using horseradish peroxidase-conjugated secondary anti-rabbit IgG antibodies (Abcam, ab6721, Cambridge, UK; 1/10,000), anti-Goat IgG antibodies (Abcam, ab6741, Cambridge, UK; 1/5000), Anti-Mouse IgG antibodies (Abcam, 97035, Cambridge, UK; 1/10,000) Membranes were then developed with the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). All membranes were stained with Coomassie blue R-350 (GE Healthcare, Buckinghamshire, UK, Falsafi et al., 2012 (link)). Arbitrary optical densities of immunoreactive bands were measured by the Image J software program (http://rsb.info.nih.gov/ij/ ). Loading controls for the BN-PAGE western blot were carried out according to Welinder and Ekblad (Welinder and Ekblad, 2011 (link)).
Coomassie blue r 350
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Coomassie Blue R-350 is a synthetic dye commonly used in biochemistry and molecular biology laboratories. It is a high-purity protein stain that binds to proteins, allowing for their visualization and quantification in various applications such as gel electrophoresis and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab75838, by Abcam (1 mentions)
Ab6741, by Abcam (1 mentions)
Ab55051, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Labscan software, by GE Healthcare (1 mentions)
Imagescanner 3 scanner, by GE Healthcare (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
Minive system, by Hoefer (1 mentions)
Phastgel blue r tablets, by GE Healthcare (1 mentions)
Brij 35, by Thermo Fisher Scientific (1 mentions)
5 protocols using coomassie blue r 350
1
Western Blot Analysis of Membrane Proteins
2
Two-step SDS-PAGE protein separation
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After IEF, strips were incubated in equilibration buffer Urea 6 M, Tris-HCl pH 8.8 75 mM, 30% (vol/vol) Glycerol, 2% (wt/vol) SDS containing 1.5% (wt/vol) DTT in the first step and 2.5% (wt/vol) iodoacetamide during the second step (15 min per step). The SDS-PAGE was carried out using 12.5% polyacrylamide gels (10 × 8 cm) on a SE 260 mini-vertical gel electrophoresis unit (Amersham Biosciences) under the following protocol: 80 V/30 min + 200 V/75 min. Then, gels were fixed with 50% (vol/vol) methanol, 10% (vol/vol) acetic acid solution, and stained with Coomassie Blue R 350 (GE Healthcare). Stained gels were scanned using an ImageScanner III scanner and LabScan software (GE Healthcare).
3
Analyzing Protein Composition via SDS-PAGE
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PLI samples were subjected to 12% SDS-PAGE, according to Laemmli, (1970) [31 (link)] under reducing by β- mercaptoethanol or non-reducing conditions. By lane, 20 μg of protein was applied. The molecular marker used was Dual Color Precision Plus, BioRad. The gels were stained using Coomassie Blue R350 (GE Healthcare).
4
SDS-PAGE Protein Separation and Analysis
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Denaturing one-dimensional electrophoresis was performed using sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis with a 12.5% concentration of acrylamide (Sigma Aldrich, USA) in the separating gel and 5% concentration in the stacking gel [25 (link),26 (link)]. Combs with a 5 mm tooth width were used to form pockets for the samples. In order to reduce disulfide bonds, the analyzed samples were placed in the sample buffer (2% lithium dodecyl sulfate (LDS), 0.065 M Tris-HCl (pH 6.8), 1% dithiothreitol (DTT), 10% glycerol, and 0.01% bromophenol blue (all from Sigma Aldrich, USA)) and were heated in a boiling water bath for 2 min. An amount of 1 μL of the Precision Plus Protein Standard (BioRad, Hercules, CA, USA) was loaded as a marker. A maximum of 40 μL (32 μL of the analyzed sample and 8 μL of the buffer) was applied to the lane. Electrophoretic separation (SDS-PAGE) of the sample proteins was performed using the Hoefer miniVE system (Hoefer Inc., Holliston, MA, USA) (gel size of 80 × 90 × 1 mm). Gels were stained with Coomassie Blue R-350 (GE Healthcare, USA) for 2 h.
5
Gelatin Zymography Analysis
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