P3/P8 mouse cortices were lysed in Pierce RIPA buffer (Thermo Scientific) containing Complete protease inhibitors (Roche) and analyzed for protein content using Bradford reagent. A total of 20 μg of protein extract was separated on a NuPAGE 3%–7% Tris-Acetate gel (Life Technologies) and blotted onto nitrocellulose. Immunoblotting was performed using Abs against Nrg1-type1 (1:1,000 dilution; ab27303, Abcam) and β-actin (1:500; ab8226, Abcam). Immunoreactive bands were detected by enhanced chemoluminescence (GE Healthcare).
Enhanced chemoluminescence
Manufactured by GE Healthcare
Enhanced chemoluminescence is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It utilizes a chemical reaction that produces light when a substrate interacts with an enzyme, allowing for the visualization and measurement of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Ab27303, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (1 mentions)
Mini protean 3 electrophoresis cell, by Bio-Rad (1 mentions)
Hybond c extra nitrocellulose membrane, by GE Healthcare (1 mentions)
Coomassie brilliant blue g 250, by GE Healthcare (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Rabbit anti β tubulin, by Cell Signaling Technology (1 mentions)
Rabbit anti mcl 1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti bcl 2, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Odyssey sa, by LI COR (1 mentions)
6 protocols using enhanced chemoluminescence
1
Nrg1 Protein Detection in Mouse Cortex
2
CD4+ T Cell Protein Analysis
3
Intestinal Brush Border Isolation
4
SDS-PAGE and Western Blot Analysis
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We performed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions (8% and 12% SDS-PAGE), according to the method of Laemmli [16 (link)]. We used a Mini-Protean 3 electrophoresis cell for the separation of proteins (Bio-Rad Laboratories, Richmond, CA, USA). Proteins were electrophoresed at 120 V, and gels stained with 0.25% Coomassie Brilliant Blue G-250 (GE Healthcare, Waukesha, WI, USA). Pre-stained standards (Fermentas, Burlington, Ontario, CA) were used for estimating the molecular weight of proteins in each sample. Following electrophoresis, proteins were transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare). We detected β-1,3-glucanosyltransferase and 1,3-β-D-glucan synthase by incubating membranes with the relevant specific murine antibodies (1 mg/mL) overnight. Membranes were then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:1000 in blocking buffer). The reaction was revealed using the peroxidase substrate ECL (Enhanced Chemo-Luminescence, GE Healthcare).
5
Protein Expression Quantification by Western Blot
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SDS PAGE and Western blotting were performed as previously described [46 (link)]. Blots were probed with the following Abs: mouse anti-ABCB1 (anti-C219 mouse monoclonal Ab; Calbiochem1:100), anti-MGMT (Millipore 1:100), rabbit anti-BCL2 (Cell Signaling clone 50E3 1:1000), rabbit anti BCL2A1 (Cell Signaling 1:1000), Rabbit anti-MCL1 (Santa Cruz 1:500) and either mouse anti-GAPDH (Sigma) or rabbit anti-β-tubulin (Cell Signalling Technology) 1:1000 as loading controls. All primary Abs were detected using goat anti-mouse/rabbit IgG HRP-linked secondary Ab (Cell Signalling Technology 1:2000) and enhanced chemoluminescence (GE Health Care Life Science) performed according to the manufacturer’s protocol.
6
Quantitative Western Blot Analysis
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Cells were lysed and proteins were collected with RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM Tris-HCl pH 7.2, 5 mM EDTA, Phosphatase and Protease Inhibitor Cocktail [Sigma-Aldrich]). Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equal amount of protein was analyzed on SDS-PAGE and transferred to polyvinylidene fluoride membranes (0.22 μm, Millipore). Membranes were then incubated at room temperature for 1 h in blocking buffer (5% low-fat milk powder in Phosphate Buffered Saline (PBS)), and after incubated overnight at 4 °C with primary antibodies. Horseradish peroxidase-conjugated secondary antibody were incubated for 2 h at room temperature. Immunoreactive bands detection was carried out using the enhancedchemoluminescence (GE Healthcare) according to the manufacturer's instructions. When indicated, IRDye secondary antibodies were used and revealed using Odyssey ® SA (Licor). We used ImageJ software to quantify western blot bands. Relative densitometry is expressed as the mean ± SD of 5 different experiments. t test: *P < 0.05, **P< 0.01, ***P< 0.001.
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