Protein concentration of cell lysates, DG‐SEC, and EV samples was measured using the Qubit Protein assay kit (Thermo Fisher Scientific) and Qubit fluorometer 3.0 following manufacturer's instructions. Cell lysates were obtained in Laemmli lysis buffer (0.125 M Tris–HCl [pH 6.8], 10% glycerol, 2.3% SDS) and proteins were lysed in reducing sample buffer (0.5 M Tris‐HCl (pH 6.8), 43% glycerol, 9.2% SDS, 5% 2‐mercaptoethanol, 5% bromophenol blue) and boiled for 5 min at 95°C. DG‐SEC sample and EV preparation lysates were obtained by mixing 30μL of sample with 5 μL of reducing sample buffer (0.5 M Tris‐HCl (pH 6.8), 43% glycerol, 9.2% SDS, 5% 2‐mercaptoethanol, 5% bromophenol blue) and boiled for 5 min at 95°C. Proteins were separated by SDS–PAGE (SDS polyacrylamide gel electrophoresis), transferred to nitrocellulose membranes, blocked in 5% non‐fat milk in PBS 0.5% Tween‐20, and incubated overnight at 4°C with primary antibodies described in the reagents section. Secondary antibodies were added for 60 min at room temperature after extensive washing with blocking buffer. After final washing, Blots were developed using the WesternBright Sirius reagent (Advansta, Menlo Park, CA) and visualized on a Proxima 2850 Imager (IsoGen Life Sciences) and images were analysed using ProXima AQ‐4 software.
Westernbright sirius reagent
Manufactured by Advansta
Sourced in United States
WesternBright Sirius reagent is a chemiluminescent substrate used for the detection of western blots. It is designed to provide a sensitive and reliable signal for the visualization of protein bands.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Glial fibrillary acidic protein (gfap), by Leica (1 mentions)
Hmgb1, by BioLegend (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
C digit scanner, by LI COR (1 mentions)
Image studio software package, by LI COR (1 mentions)
4 protocols using westernbright sirius reagent
1
Quantifying Protein Levels Across Samples
2
Western Blot Analysis of Circadian Clock Proteins
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The proteins were loaded onto a 10% SDS-PAGE gel and run at 100 V for 2 h. Once the migration was completed, the proteins were transferred to Hybond® ECL™ nitrocellulose membranes for 1 h 30 min using a semidry blotting machine. The membrane was subsequently washed with 1× TBS 1×/0.1% Tween 20 (TBS-T) and blocked with 5% skim milk in TBST for 1 h. After washing, the membrane was stained with the appropriate primary antibody overnight. The antibodies against all the clock factors were described [52 (link)] (final dilution 1:1000). CDK5 was purchased from Cell Signaling Tech (1:1000), tubulin from Abcam (1:1000), and Lamin B from Santa Cruz (1:1000). The day after, membranes were washed three times with TBST followed by incubation with an HRP-conjugated secondary antibody for 1 h at room temperature and three more washing steps in TBST. HRP activity was detected with the WesternBright Sirius reagent (Advansta Inc., San Jose, CA, USA). The luminescence signal was digitally acquired with an Azure Biosystem Imager.
3
Western Blot Analysis of Protein Extracts
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Total protein extracts from slices were obtained by lysing slices in RIPA buffer, followed by sonication and centrifugation at 12,000 g for 10 min. Total protein concentrations were measured using Nanodrop ND-100 Spectrophotometer, and Western blot was carried out as usual in our lab (Santos et al., 2018 (link)). The primary antibodies were as follows: NG2 (rabbit, 1:250, Merck Millipore), MBP (rat, 1:250, Serotec), S100B (rabbit, 1:500, Abcam), RAGE (rabbit, 1:800, Abcam), GFAP (rabbit, 1:500, NovoCastra), high-mobility group box 1 (HMGB1, mouse, 1:200, BioLegend), phosphorilated nuclear factor-κB (pNF-κB rabbit, 1:500, Abcam), NF-κB (rabbit, 1:500, Santa Cruz Biotechnology), or β-actin (mouse, 1:10,000; Sigma). Protein bands were detected using WesternBright Sirius reagent (Advansta, Menlo Park, CA, USA) and visualized using ChemiDocTM XRS System (Bio-Rad, Hercules, CA, USA). Results were normalized to β-actin expression for each experiment.
4
Cytokine Profile Analysis of Conditioned Media
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Conditioned media was analyzed using the Proteome Pro ler Human Cytokine Array (R&D Systems) as directed by the manufacturer. The arrays were developed using WesternBright Sirius reagent (Advansta, San Jose, California, USA) on a C-Digit scanner (LI-COR, Lincoln, Nebraska, USA) and signal densities were determined using Image Studio software package (LI-COR). Data were normalized to CSDS strain pro le for each paired ex run.
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