Cells were lysed with SDS lysis buffer. Cell lysates (10 μg of proteins) were separated by SDS-PAGE, electrophoretically transferred to a PVDF membrane (Immobilon; Millipore) and incubated with Blocking One (Nacalai) and the indicated antibodies diluted with Can Get Signal buffer (TOYOBO). Immunoreactive bands were detected with an enhanced chemiluminescence (ECL) detection system (ECL Western Blotting Detection Reagents; GE Healthcare, Pierce™ ECL Plus Western Blotting Substrate; Thermo Fisher Scientific) and visualized with Amersham Hyperfilm ECL film (Amersham).
Can get signal buffer
Manufactured by Toyobo
Sourced in Japan
The Can Get Signal buffer is a device designed to condition and amplify electrical signals. It functions by receiving an input signal and providing a stable, amplified output signal. The core purpose of this product is to optimize signal transmission without interpretation of its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Lumi light plus kit, by Roche (3 mentions)
Proteinase inhibitor cocktail, by Merck Group (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Phosstop, by Roche (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Immobilon, by Merck Group (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Amersham hyperfilm ecl film, by Cytiva (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Phosphatase inhibitor tablet, by Roche (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using can get signal buffer
1
Western Blotting Protein Detection Protocol
2
Protein Extraction and Western Blot Analysis
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Cells were harvested from 6-well plates. For the extraction of protein in tissues, excised organs were homogenized in NP-40 lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor tablet (PhosStop, Roche). The lysate was centrifuged for 10 min at 15000 x g (maximum) at 4 °C, and the supernatant was then collected. The protein concentration was determined by BCA protein assay (Thermo SCIENTIFIC, Waltham, MA) with bovine serum albumin (BSA) standard. Equal amounts of protein samples were loaded, separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (PVDF, Immobilon-P, Millipore). The membranes were blocked in PBS–Tween (0.1%) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5% nonfat dried milk or Can Get Signal Buffer (TOYOBO, Japan). The bands were detected using Lumi-light Plus kit (Roche) and LAS-3000. Band intensities were quantified with the NIH Image J software. All primary antibodies were used at a dilution of 1:1000.
3
Protein Extraction from Tissues
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For protein extraction from tissues, excised organs were homogenized in NP-40 lysis buffer (50 mM Tris-HCl, pH8.0, 150 mM NaCl, 5 mM EDTA, and 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor tablet (Roche, Penzberg, Germany). The lysate was centrifuged for 10 minutes at 15000 × g (maximum) and 4 °C, and the supernatant was then collected. The protein concentration was determined with a BCA protein assay (Thermo Scientific, Waltham, MA, USA), with bovine serum albumin (BSA) as the standard. Equal amounts of protein samples were loaded into SDS-polyacrylamide gels for electrophoresis, and then, the separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerrica, MA, USA). The membranes were blocked in PBS–Tween (0.1%) (PBST) with 1% BSA or 5% nonfat dried milk and were then probed with the primary antibodies diluted PBST-BSA, 5% nonfat dried milk or Can Get Signal Buffer (TOYOBO, Osaka, Japan). The bands were detected using a Lumi-light Plus kit (Roche, Penzberg, Germany) and LAS-3000. All primary antibodies were used at a dilution of 1:1000.
4
Western Blot Analysis of hiMGLs and hiPSCs
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hiMGLs and hiPSCs were lysed in cold RIPA buffer. Complete® Protease Inhibitor and Phosphatase Inhibitor were added freshly to RIPA buffer directly before use. Cell lysates were homogenized by passing through a 1-ml syringe, followed by centrifugation for 30 min at 15,000 rpm at 4 °C. Supernatants were collected and stored at − 80 °C. Protein concentration was determined using a BCA Protein Assay Kit. Ten micrograms of total protein was loaded and separated by 10–20% Extra PAGE gels (Nacalaitesque, Japan) in SDS running buffer. Gels were transferred to nitrocellulose membranes using a Trans-Blot Turbo system. The membranes were blocked with 5% milk in TBST for an hour at room temperature, followed by incubation with α-tubulin antibody or DAP12 antibody at 4 °C overnight. The next day, the membranes were incubated with the corresponding Odyssey secondary antibody diluted in TBST and CanGetSignal buffer (TOYOBO, Japan) (1:1) for an hour at room temperature. Images were captured and analyzed using the Odyssey Imaging system (LI-COR Biosciences, USA).
5
Western Blot Analysis of Cell Lysates
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Cell lysate samples were prepared in NP-40 lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (PhosStop, Roche) or by direct lysis in boiling Laemmli SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 25% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue). Protein samples were electrophoresed on 10% SDS-polyacrylamide gel and transferred to polyvinylidine difluoride membranes (Immobilon-P, Millipore). The membranes were blocked in Tris-buffered saline (TBS)-Tween (0.1%) with either 5% bovine serum albumin (BSA) or 5% nonfat dried milk and were then incubated with each primary antibody diluted in TBS containing 0.1% Tween 20 supplemented with either 5% nonfat dried milk or 5% BSA or in the Can Get Signal buffer (TOYOBO). Bound antibody was detected by a Lumi-light Plus kit according to the manufacturer’s instructions (Roche Diagnostics). Band intensities on blots were quantified using NIH Image J software. All primary antibodies were used at a dilution of 1:1,000, except anti-β-actin (1:5,000) and anti-NDRG2 (E20, 1:250). Representative full-gel bots are provided in the Supplementary Fig. 22 )
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