Pellets from SIN-1 treated cells (2x106 cells) were dissolved in Laemmli sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 50 mmol/L DTT, 2% w/v SDS, 20% v/v glycerol, 0.2% w/v bromophenol blue), sonicated (8 s, 30% amplitude) and boiled (5 min, 95 °C). Proteins were separated by SDS-PAGE (15%-Tris-Tricine-Gel; BioRad) and blotted onto a PVDF membrane (0.2 µm GE Healthcare, Buckinghamshire, England; Trans-Blot® Cell, BioRad). Membranes were blocked overnight (4 °C, 5% dry milk in TPBS), incubated with primary mouse monoclonal anti-LC3 antibody (3 h, 1:250 in blocking buffer, RT) and washed (TPBS). Peroxidase-conjugated goat anti-mouse antibody (1 h, 1:10,000 in blocking buffer, RT) was added. Membranes were washed and developed using Western Lightning Plus (Perkin Elmer, Waltham, USA) and Lucent Blue X-ray film (Advansta, Menlo Park, CA, USA). Images were quantified using ImageJ.
Lucent blue x ray film
Manufactured by Advansta
Sourced in United States
Lucent Blue X-ray film is a photographic film designed for use in X-ray imaging applications. It is a high-quality film that provides clear and detailed images of X-ray exposures. The film is sensitive to X-ray radiation, allowing it to capture and record the image information for later analysis and interpretation by medical professionals or researchers.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Westernbright ecl, by Advansta (2 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Non fat milk, by Merck Group (2 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Runblue precast gradient gels, by Abcam (2 mentions)
Supersignal west femto max sensitivity ecl, by Advansta (2 mentions)
Immobilon p pvdf, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by GE Healthcare (2 mentions)
Western lightning plus, by PerkinElmer (1 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Foxo3, by Cell Signaling Technology (1 mentions)
Neuronal protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Casp3, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Westernbright ecl hrp substrate, by Advansta (1 mentions)
8 protocols using lucent blue x ray film
1
Western Blot Analysis of LC3 Protein
2
Western Blot Analysis of Neuronal Proteins
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Total protein was extracted using Neuronal Protein Extraction Reagent (ThermoFisher Scientific), and proteins were separated by electrophoresis on a 12% polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were incubated overnight with the primary antibodies at 4 °C with optimal dilution following the manufacturer’s recommendations, and then incubated with the appropriate secondary antibody at a 1:2000 dilution for 1 h at room temperature. The FOXO3 (Cat No. 2497, Cell Signaling Technology, Danvers, MA, USA), p-FOXO3 (Thr32) (Cat No. 9464S, Cell Signaling Technology), p-AKT (Ser473) (Cat No. 4060, Cell Signaling Technology), BCL2 (Cat No. 7382, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CASP3 (Cat No. 7184, Santa Cruz Biotechnology), PTEN (Cat No. 9188, Cell Signaling Technology), and ACTIN β (Cat No. 4967, Cell Signaling Technology) primary antibodies were used in the Western blot experiments. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000; Cell Signaling Technology) for 1 h at room temperature. The specific signals were detected with enhanced chemiluminescence (Western Bright ECL HRP substrate; Advansta, Menlo Park, CA, USA) and Lucent Blue X-ray film (Advansta).
3
Fasciola Protein Purity and Immunogenicity
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The purity of the protein was detected by 12% SDS-PAGE, followed by Coomassie blue staining. Also, 20 μg of the purified rFgRab10 protein were resolved on 12% SDS-PAGE and transferred onto Hybond-C extra nitrocellulose membrane (Amersham, London, UK). The membrane was washed 5 times (5 min each) in TBS-T. The membrane was then incubated with primary antibodies (serum from sheep naturally infected with Fasciola) for 1 h at 37 °C (1:100 in TBS-T). After washing 5 times in TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-goat immunoglobulin (Ig)-G antibody (Sigma, St. Louis, MO, USA) at 37 °C for 1 h (1:2000 in TBS-T). The immunoreaction was visualized using WesternBright ECL (Advansta, Menlo Park, CA, USA) and LucentBlue X-ray film (Advansta, Menlo Park, CA, USA).
4
Immunoblotting of Yeast Protein Extracts
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For immunoblotting, yeast cell extracts were resuspended at identical cell densities in sodium dodecyl sulphate (SDS) loading dye and lysed by boiling for 6 min and vortexing for 5 min with glass beads. Protein samples were separated into 7.5–10% SDS-PAGE gels and transferred to nitrocellulose membranes (Hybond-C, GE Healthcare).
The primary antibodies used were raised against yeast Tim22p (1:500, sc-14042, Santa Cruz Biotechnology, Dallas, TX, USA), yeast Cox2p (1:6000, 4B12A5, ThermoFisher Scientific, Waltham, MA, USA), yeast Cytochrome c (1:10,000, Davids Biotechnologie, Regensburg, Germany), yeast Pgk1p (1:30,000, 22C5D8, ThermoFisher Scientific, Waltham, MA, USA), HA (1:1000, Y-p11, Santa Cruz Biotechnology, Dallas, TX, USA) and c-Myc (1:1000, ThermoFisher Scientific, Waltham, MA, USA).
Secondary antibodies used were anti-goat IgG-HRP (1:5000), anti-mouse IgG-HRP (1:10,000, Molecular probes, Eugene, OR, USA) and anti-rabbit IgG-HRP (1:10,000, Sigma, St. Louis, MO, USA).
Membranes were incubated with WesternBright ECL (Advansta, San Jose, CA, USA), exposed to LucentBlue X-ray film (Advansta), scanned on a Molecular Imager GS900, and quantified using Image Lab Software version 6.1 (Bio-Rad, Hercules, CA, USA).
Full-length blots corresponding to the blots displayed in various figures and used for data quantification are provided inFigures S4–S6 .
The primary antibodies used were raised against yeast Tim22p (1:500, sc-14042, Santa Cruz Biotechnology, Dallas, TX, USA), yeast Cox2p (1:6000, 4B12A5, ThermoFisher Scientific, Waltham, MA, USA), yeast Cytochrome c (1:10,000, Davids Biotechnologie, Regensburg, Germany), yeast Pgk1p (1:30,000, 22C5D8, ThermoFisher Scientific, Waltham, MA, USA), HA (1:1000, Y-p11, Santa Cruz Biotechnology, Dallas, TX, USA) and c-Myc (1:1000, ThermoFisher Scientific, Waltham, MA, USA).
Secondary antibodies used were anti-goat IgG-HRP (1:5000), anti-mouse IgG-HRP (1:10,000, Molecular probes, Eugene, OR, USA) and anti-rabbit IgG-HRP (1:10,000, Sigma, St. Louis, MO, USA).
Membranes were incubated with WesternBright ECL (Advansta, San Jose, CA, USA), exposed to LucentBlue X-ray film (Advansta), scanned on a Molecular Imager GS900, and quantified using Image Lab Software version 6.1 (Bio-Rad, Hercules, CA, USA).
Full-length blots corresponding to the blots displayed in various figures and used for data quantification are provided in
5
Quantitative Western Blot Analysis Protocol
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Collection of total cellular protein, electrophoresis and blotting were performed as described previously.23 (link) Membranes were blocked for 1 h in Tris-buffered saline supplemented with 0.05% Tween 20 and either 5% non-fat milk (Sigma) or 5% bovine serum albumin (GE Healthcare, Little Chalfont, UK). Subsequently, membranes were incubated overnight at 4 °C with primary antibodies. Detailed information on used antibodies is provided in Supplementary Table S2 . After washing (3 × Tris-buffered saline supplemented with 0.05% Tween 20 for 10 min), membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature, followed by washing as described above. Signals were detected by chemiluminescence using ECL Plus (Thermo Scientific, Waltham, MA, USA) and Lucent Blue X-ray films (Advansta, Menlo Park, CA, USA). Stripping of blots was performed using Restore Plus Western Blot Stripping Buffer (Thermo Scientific) according to the manufacturer's instructions, followed by re-blocking in Tris-buffered saline supplemented with 0.05% Tween 20 with 5% milk for 1 h. Detection of β-tubulin was performed as loading control. Densitometric analysis of bands was performed using ImageJ (http://imagej.nih.gov ).
6
Western Blot Protein Analysis Protocol
7
Protein Separation and Detection using BioRad and RunBlue Gels
8
Protein Separation and Detection Protocol
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For protein separation and detection, the BioRad system, and RunBlue precast gradient gels (expedeon) were used. Prior to loading on the gel, samples were diluted in Laemmli buffer (Laemmli 1970 ) containing 100 mM DTT and heated to 98°C for 10 min. Samples were spun down and loaded using a Hamilton syringe. SDS-PAGE was performed at a constant voltage of 150V for 50 min. For Western Blot, Immobilon-P PVDF or nitrocellulose membranes were used in a BioRad system (100V for 90 min while keeping the system cool). Subsequently, membranes were blocked in 5% milk in TBST for 1h and incubated in primary antibody diluted 1:1000 (see Table S14 for manufacturer and origin and antibody dilutions used) overnight. Membranes were washed 3 times for 5 min in TBST and subsequently incubated in secondary antibody conjugated to HRP (see Table S14) for 1h in 5% milk in TBST. After washing 3 times for 5 min, exposure using SuperSignalTM West Pico Plus chemiluminescent substrate (Thermo scientific) or Supersignal West Femto Max Sensitivity ECL onto Lucent Blue X-Ray films (advansta) or Kodak film in the dark was used to detect protein bands.
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