Neuronal cultures were collected and lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris/HCl (pH 8.0), with phosphatase and protease inhibitors cocktail) on ice. Lysates were vortexed and cleared by centrifugation (14000 g, 10 min, 4°C). Supernatants were collected, and protein concentrations were determined by the Bicinchoninic Acid Protein Assay Kit (Thermo Scientific), according to the manufacturer's protocol. Samples were analyzed by SDS/PAGE (4-12% gradient gels), and proteins were transferred on to nitrocellulose membranes using the iBlot2 system (Life Technologies). Membranes were blocked with 5% (w/v) non-fat dried skimmed milk for 1 h at room tempera-ture, and they were incubated with anti-BRCA1 and anti-actin, as a loading control, overnight at 4°C. Membranes were then washed with TBS (Tris-buffered saline; 10 mM Tris/HCl and 150 mM NaCl (pH 7.4)) and incubated with anti-rabbit-HRP and anti-mouse-HRP for 1 h at room temperature. Chemiluminescent signal was visualized with Prometheus ProSignal Pico (Genesee Scientific) on Blue Devil autoradiography films (Genesee Scientific).
Blue devil autoradiography film
Manufactured by Genesee Scientific
Blue Devil Autoradiography film is a photographic film used to detect and visualize radioactive signals in biological samples. It is designed for autoradiography applications, which involve exposing the film to radioactive materials to produce images of the radioactive patterns. The film provides a reliable and accurate method for detecting and analyzing radioactive markers in experiments.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Merck Group (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Prometheus prosignal pico, by Genesee Scientific (3 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Iblot2 system, by Thermo Fisher Scientific (2 mentions)
Anti fasn, by Proteintech (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti bckdha, by Novus Biologicals (2 mentions)
Anti crat, by Novus Biologicals (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (2 mentions)
Nbp1 79616, by Cell Signaling Technology (2 mentions)
Precision plus protein standard, by Bio-Rad (1 mentions)
Trans blot turbo device, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
8 protocols using blue devil autoradiography film
1
BRCA1 Protein Expression Analysis
2
Immunoblot Analysis of TagRFP
3
Western Blot Analysis of Autophagy Proteins
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Neuronal cultures were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris/HCl (pH 8.0), with phosphatase and protease inhibitors cocktail) on ice. Lysates were vortexed and cleared by centrifugation (14000 g, 10 min, 4°C). Supernatants were collected, and protein concentrations were determined by the Bicinchoninic Acid Protein Assay Kit (Thermo Scientific). Samples were analyzed by SDS/PAGE (4–12% gradient gels), and proteins were transferred on to nitrocellulose membranes using the iBlot2 system (Life Technologies). Membranes were blocked with 5% skimmed milk for 1 hr at room temperature, and they were incubated with the primary antibodies (anti-LC3, Htt, anti-actin or anti-ATG7) overnight at 4°C. Membranes were then washed with TBS (Tris-buffered saline; 10 mM Tris/HCl and 150 mM NaCl (pH 7.4)) and incubated with anti-rabbit-HRP or anti-mouse-HRP for 1 hr at room temperature. Chemiluminescent signal was visualized with Prometheus ProSignal Pico (Genesee Scientific) on Blue Devil autoradiography films (Genesee Scientific).
4
Quantification of Adipocyte Proteins
5
Western Blot Analysis of EV Markers
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1 × 106 EVs for each condition shown in Supplementary Fig. 1B were processed for Western blot analysis as follows. iEVs were counted by NTA and lysed at 70 °C for 5 minutes in 5 × loading dye buffer in a total volume of 20 µL. 15 µL samples were loaded into a 4–20% mini protean TGX gel (Bio-Rad). Precision Plus Protein Standard was used as a marker for weight (Bio-Rad). Gel was ran at 100 volts for 75 minutes and then transferred to 0.2 µm PVDF membrane using a Trans Blot Turbo device (Bio-Rad). Wester blotting protocol from Cell Signal Inc. was followed for antibody detection. Primary antibodies rat anti-mouse CD63 monoclonal antibody (Cat#564222, BD Biosciences) and hamster anti-mouse CD81 monoclonal antibody (Cat#559519) were both used at 1:1,000 dilution. Horseradish peroxidase-labeled rabbit antibodies to rat/hamster IgG were used at 1:2,500 dilution. Bound antibodies were revealed with Clarity Western ECL substrate (Bio-Rad). Blot was exposed for 10 minutes with Blue Devil autoradiography film (Genesee Scientific).
6
Quantification of Adipocyte Proteins
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3T3L1 adipocytes or 100–150 mgs of ground eWAT were lysed in ice-cold RIPA buffer with 1× protease inhibitor (Sigma-Aldrich). 30 μg of total protein was separated on a 12% SDS-PAGE gel for BCKDH blots or 20 ugs of total protein was separated on a 4–20% SDS-PAGE gel (mini-protean TGX gels, Bio-Rad) for CrAT and FASN blots. The proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-Bckdha (Novus Biologicals NBP1–79616) (1:1,000 dilution), anti-Beta-Actin (Cell Signaling 4970S) (1:5,000), anti-GAPDH (Cell signaling 5174S), anti-CrAT (Novus NBP2–15999), anti-FASN (Proteintech 10624–2-AP). Specific signal was detected with horseradish peroxidase-conjugated secondary antibody goat anti-rabbit (1:2,500 – 1:10,000) using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and developed using Blue Devil Autoradiography film (Genesee Scientific) or BioRad Chemidoc XRS+.
7
Western Blot Analysis of Bckdha in 3T3-L1 Adipocytes
8
Western Blot Analysis of Bckdha in 3T3-L1 Adipocytes
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3T3–L1 adipocytes with Control KD or Bckdha KD were lysed in ice-cold RIPA buffer with 1x protease inhibitor (Sigma-Aldrich. 30 μg of total protein was separated on 12% SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and immunoblotted with rabbit anti-Bckdha (Novus Biologicals NBP1-79616) (1:1000 dilution) and mouse anti-Beta-Actin (Cell Signaling 8H10D10) (1:5,000). Specific signal was detected with horseradish peroxidase-conjugated secondary antibody goat anti-rabbit (1:2,500) or rabbit anti-mouse (1:10,000) using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and developed using Blue Devil Autoradiography film (Genesee Scientific).
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