Expression of the enzymes involved in glucose metabolism and in cytosolic and ER PPP was determined by Western blot, using standard procedures. Anterior and posterior brain regions were homogenized in PBS in the presence of a protease inhibitor cocktail for mammalian cells, and total protein was measured by Bradford assay. After SDS-PAGE, performed according to the standard method on 10% precast gels (BioRad), proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with Tris Buffered Saline (TBS) plus 0.15% Tween 20 (TBSt) containing 5% nonfat dry milk and incubated overnight at 4 °C with the following rabbit polyclonal antibodies: anti-HK II (1:1000, Cell Signalling #2867), anti-G6Pase (1:1000, Abcam, ab93857), anti-PFKP (1:200, Cell Signalling #8164), anti-G6PD (1:1000, Abcam, ab210702), anti_H6PD (1:1000, Abcam, ab170895), or anti-actin (1:10,000, Thermo-Fisher MA5-11869). After washing with TBSt, the membrane was incubated with an anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (BioRad) and developed with Clarity Western ECL Substrate (BioRad). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.
Alliance 6.7 wl 20m
Manufactured by Uvitec
Sourced in United Kingdom
The Alliance 6.7 WL 20M is a laboratory instrument designed for analytical applications. It features a 20-megapixel camera and supports a wide range of wavelengths. The core function of this product is to provide high-resolution imaging capabilities for various scientific and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Uv1d software, by Uvitec (6 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (5 mentions)
Anti actin, by Thermo Fisher Scientific (4 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Gradient gel, by Bio-Rad (3 mentions)
A0168, by Merck Group (2 mentions)
Liteablot plus, by Euroclone (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti drp1, by Abcam (2 mentions)
Precast gel, by Bio-Rad (2 mentions)
Anti hkii, by Cell Signaling Technology (1 mentions)
Anti g6pase, by Abcam (1 mentions)
Anti pfkp, by Cell Signaling Technology (1 mentions)
Anti rabbit igg antibody, by Bio-Rad (1 mentions)
Protease inhibitor cocktail for mammalian cells, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti cdk1, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β tubulin 3, by Merck Group (1 mentions)
11 protocols using alliance 6.7 wl 20m
1
Glucose Metabolism Enzyme Expression Analysis
2
Protein Expression Analysis by Western Blot
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Expression of CLUH, PGC-1α, and SIRT6 proteins was determined by Western blot (WB), using standard procedures. MNCs were lysed using lysis buffer (150 mM NaCl, 20 mM TRIS-HCl pH 7.4, 2 mM EDTA, and 1% NP40) containing a protease inhibitor cocktail for mammalian cells (Sigma) and total protein was measured by Bradford assay [40 (link)]. After SDS-PAGE, performed according to the standard method on 4–20% polyacrylamide gels, proteins were transferred to a nitrocellulose membrane (BioRad Laboratories). The membrane was blocked for 1 h with PBS-0.1% Tween 20 (PBSt) containing 5% non-fat dry milk and incubated overnight at 4 °C with the following primary antibodies: anti-CLUH (1:1000, cod: A301-764A, Bethyl Lab), anti-PGC-1α (1:1000, cod: ab77210, Abcam), and anti-SIRT6 (1:1000, cod: ab88494, Abcam).
After washing with PBSt, the membrane was incubated with an anti-rabbit or anti-mouse IgG antibody conjugated with horseradish peroxidase (BioRad Laboratories) and developed with Clarity Western ECL Substrate (BioRad Laboratories). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin level in the same membrane.
After washing with PBSt, the membrane was incubated with an anti-rabbit or anti-mouse IgG antibody conjugated with horseradish peroxidase (BioRad Laboratories) and developed with Clarity Western ECL Substrate (BioRad Laboratories). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin level in the same membrane.
3
Analyzing DNA Damage via SDS-PAGE
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Denaturing electrophoresis (SDS-PAGE) was performed on 4–20% gradient gels (Bi- oRad) (Hercules, CA, USA), loading 30 μg of protein homogenate for each sample. After the electrophoresis separation, proteins were transferred onto the nitrocellulose membrane, which was blocked with 5% BSA. Membrane was incubated with a mouse antibody against phosphorylated-γ-H2AX (Merck Millipore, cod: 05-636), a marker of DNA double-strand breaks, and a mouse antibody against β-actin (Santa Cruz Biotechnology, cod: sc-1616), used as housekeping protein. All primary antibodies were diluted 1:10,000 in PBS plus 0.15% tween (PBSt). Bands were detected and analyzed for optical density using an enhanced chemiluminescence substrate (ECL, BioRad), a chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC), and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.
4
SDS-PAGE and Immunoblotting for Protein Analysis
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We loaded 30 μg of proteins into a 4–20% gradient gel (#4561094, BioRad, Hercules, CA, USA) to perform a denaturing electrophoresis (SDS-PAGE). As primary antibodies, we employed anti-p53 (#9282, Cell Signaling Technology, Danvers, MA, USA), anti-CDK1 (#MA1-19057, Thermo Fisher Scientific, Waltham, MA, USA), and anti-Actin (#MA1-140, Thermo Fisher Scientific, Waltham, MA, USA), all diluted in PBS plus 0.15% Tween 20 (PBSt; #11332465001, Roche, Basel, Switzerland). A0168 and SAB3700870, both from Merck, Darmstadt, Germany, were used as specific secondary antibodies, and they were all diluted 1:10,000 in PBSt. Using a chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and an enhanced chemiluminescence substrate (ECL, # 1705061, BioRad, Hercules, CA, USA), bands were detected, and the signal intensity was measured by Uvitec-1D 2015 software (UVITEC, Cambridge, UK). The actin signal was used to normalize each band of interest.
5
Spinal Cord Protein Expression in ALS Mice
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Spinal cord synaptosomal or total tissue proteins from 30-, 60-, and 90-day-old WT and SOD1G93A mice were separated by means of SDS-polyacrylamide gel electrophoresis; 4% to 20% gradient gels were used. The concentration of proteins in each sample fell in the linear portion of the standard curve. Proteins were transferred to nitrocellulose membranes and electroblotted proteins were monitored using Naphthol blue black staining (Sigma Aldrich, MO, USA). Membranes were saturated in 5% skimmed-milk solution and incubated with the following antibodies: Mouse monoclonal anti-mGluR1 (1:500, cat n. 610964; BD Biosciences, San Jose, CA, USA); rabbit monoclonal anti-mGluR5 (1:500, cat n. ab53090; Abcam, Cambridge, UK); mouse monoclonal anti-glyceraldeide phosphate dehydrogenase, GAPDH (1:10000, cat. N. G8795; Sigma Aldrich, MO, USA); mouse monoclonal anti-β-tubulin III (1:1000; cat. N. T8578; Sigma-Aldrich, MO, USA). After incubation with peroxidase-coupled secondary antibodies, protein bands were detected and analyzed for optical density using an enhanced chemiluminescence substrate (ECL, LiteAblot PLUS, Euroclone, Milan, Italy) and a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC, Cambridge, UK), and UV1D software (UVITEC). Bands of interest were normalized for β-tubulin III or GAPDH levels in the same membrane.
6
Quantifying Mitochondrial Protein Levels
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After denaturing electrophoresis (SDS-PAGE), nitrocellulose membrane was incubated with anti-MFN2 (#11925S, Cell Signaling Technology, Danvers, MA, USA), anti-DRP1 (#ab184247, Abcam, Cambridge, UK), anti-UCP2 (#sc-6525, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-Actin (#sc-1616, Santa Cruz Biotechnology, Dallas, TX, USA). All primary antibodies were diluted in PBS plus 0.15% Tween (PBSt, Tween was from Roche, Basilea, Switzerland, #11332465001). Secondary antibodies (#A0168 and #SAB3700870, Sigma-Aldrich, St. Louis, MO, USA) were diluted 1:10,000 in PBSt. Bands were evaluated by a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC., Cambridge, UK) using an enhanced chemiluminescence substrate (ECL, #1705061, BioRad, Hercules, CA, USA). Band intensity was evaluated by AllianceTM Q9-Series software (UVITEC, Cambridge, UK). All the signals were normalized versus the actin signal.
7
Western Blot Analysis of Mitochondrial Proteins
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Denaturing electrophoresis (SDS-PAGE) was performed on 30 μg of proteins employing a 4–20% gradient gel (#4561094, BioRad, Hercules, CA, USA). The following primary antibodies were used: anti-SRSF4 (#PA5-36366, ThermoFisher Scientific, Waltham, MA, USA), anti-phospho-mammalian target of rapamycin (mTOR, #5536S, Cell Signaling Technology, Danvers, MA, USA), anti-mTOR (#2983S, Cell Signaling Technology, Danvers, MA, USA), anti-clustered mitochondria homolog (CLUH, #A301-764A, Bethyl Laboratories, Montgomery, TX, USA), anti-OPA1 (#HPA036926, Merck, Darmstadt, Germany), anti-DRP1 (#ab184247, Abcam, Cambridge, UK), and anti-Actin (#MA1-140, ThermoFisher Scientific, Waltham, MA, USA). All primary antibodies were diluted following the manufacturer’s instructions in PBS plus 0.15% Tween 20 (PBSt; #11332465001, Roche, Basel, Switzerland). Specific secondary antibodies were employed (#A0168 and #SAB3700870, Merck, Darmstadt, Germany), all diluted 1:10,000 in PBSt. Bands were detected in the presence of an enhanced chemiluminescence substrate (ECL, #1705061, BioRad, Hercules, CA, USA) using a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC, Cambridge, UK). Band intensity was evaluated with Uvitec-1D 2015 software (UVITEC, Cambridge, UK). All bands of interest were normalized versus the actin signal detected on the same membrane.
8
Western Blot Analysis of Apoptotic Markers
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Denaturing electrophoresis (SDS-PAGE) was performed on 4–20% gradient gels (BioRad). In total, 30 μg of proteins were loaded for each sample. The following antibodies were employed: anti-Bcl2 (Cell Signaling, #15071), anti-BAX (MilliporeSigma, cod: 06–449), anti-phospho-p53 (Ser 15) (Santa Cruz Biotechnology, cod: sc-101762), anti-p53 (Santa Cruz Biotechnology, cod: sc-98), and anti-Actin (Santa Cruz Biotechnology, cod: sc-1616). All primary antibodies were diluted 1:1000 in PBS plus 0.15% tween (PBST). Specific secondary antibodies were employed (Sigma-Aldrich), diluted 1:10,000 in PBST. Bands were detected and analyzed for optical density using an enhanced chemiluminescence substrate (ECL, BioRad), a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC), and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.
9
Western Blot Analysis of Apoptosis Regulators
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Expression of Mcl-1 and Bcl-xL was determined by Western blot, using standard procedures. Quiescent and activated CLL cells treated or not with 20 μM BRB were lysed in the presence of a protease inhibitor cocktail for mammalian cells and total protein was measured by Bradford assay. After SDS-PAGE, performed according to the standard method on 4–20% precast gels (BioRad), proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with Tris Buffered Saline (TBS) plus 0.15% Tween 20 (TBSt) containing 5% non-fat dry milk and incubated over-night at 4 °C with the following mouse monoclonal antibodies: anti-Mcl-1 (1:500, Santa-Cruz Biotechnology, sc-12756), anti-Bcl-xL (1:500, Santa-Cruz Biotechnology, sc-8392) or anti-actin (1:10,000, Thermo-Fisher MA5-11869). After washing with TBSt, the membrane was incubated with an anti-mouse IgG antibody conjugated with horse radish peroxidase (HRP) (BioRad) and developed with Clarity Western ECL Substrate (BioRad). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin level in the same membrane.
10
Mitochondrial Protein Expression in SOD1 ALS Mouse Model
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Mouse spinal cord synaptosomes and gliosomes from 30, 60, 90, and 120 days-old wtSOD1 and mice SOD1 G93A mice were lysed and used for Western blot analyses. Protein extracts (20 µg) were loaded onto 8-20% gradient polyacrylamide gel (BioRad, Hercules, California, USA), separated by SDS-PAGE, and transferred to nitrocellulose membranes. Non-specific membrane binding sites were saturated in 5% skimmed-milk solution and membranes were incubated over-night at 4 °C with the following primary antibodies: rabbit anti-ATP5B 1:500 (a subunit of F1 moiety of ATP synthase, Sigma Aldrich, Saint Louis, Missouri, USA), antiMT-CO2 (a subunit of respiratory complex IV, Abcam, Cambridge, UK), anti-mitochondrial import inner membrane translocase, subunit8A (TIM, Santa Cruz Biotechnology, California, USA) and mouse anti-Gapdh 1:5000 (Sigma Aldrich, Saint Louis, Missouri, USA). After washing, membranes were incubated for 60 min at room temperature with the following peroxidase-coupled secondary antibodies: goat anti-rabbit 1:2000 (Sigma Aldrich, Saint Luis, Missouri, USA). Bands were detected and analyzed for optical density using an enhanced chemiluminescence substrate (ECL, LiteAblot PLUS, Euroclone, Milan, Italy) and a chemiluminescence system (Alliance 6.7 WL 20M, UVITEC, Cambridge, UK), and UV1D software (UVITEC). Bands of interest were normalized for Gapdh level in the same membrane.
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