Cell culture suspensions were used for total protein isolation. Cells were incubated separately with either 1 or 2 μM IM, 4 mM DCA or in combination with these two agents for 72 h. Cell suspensions were harvested at a 60-80% confluency, washed twice with cold PBS (1X), centrifuged at 1,100 × g and the resulting pellets were lysed with RIPA buffer (1 M sodium phosphate, 5 M NaCl, 10% SDS, 10% NP-40 and 1% deoxycholic acid sodium salt, pH 7.2), supplemented with protease inhibitors cocktail (1X) (Sigma-Aldrich; Merck KGaA). After centrifugation at 10,000 × g for 15 min at 4°C, all supernatants were collected, aliquoted and stored at −20°C, as representative of the cellular total protein content. Untreated cell cultures incubated without any agent served as control experiments.
Cocktail protease inhibitors 1x
Manufactured by Merck Group
Cocktail protease inhibitors/1X is a laboratory product that contains a mixture of protease inhibitors. Protease inhibitors are chemical compounds used to inhibit the activity of proteases, which are enzymes that break down proteins. This product is designed for use in research and scientific applications where the controlled inhibition of protease activity is required.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Gp91phox, by BD (3 mentions)
α tubulin, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Fortis Life Sciences (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Visionworks ls image acquisition and analysis software, by Analytik Jena (2 mentions)
Quantikine colorimetric sandwich elisa, by R&D Systems (2 mentions)
Chemidoc it imaging system, by Analytik Jena (2 mentions)
Glu tubulin, by Abcam (1 mentions)
Chemi doctm xrs 2015, by Bio-Rad (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
P44 p42 mapk, by Cell Signaling Technology (1 mentions)
Phospho akt thr 308, by Merck Group (1 mentions)
Hdac4, by Cell Signaling Technology (1 mentions)
Nf kb, by Cell Signaling Technology (1 mentions)
Hdac2, by Santa Cruz Biotechnology (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Chemi doc xrs 2015, by Bio-Rad (1 mentions)
Criterion tgx stain free precast gel, by Bio-Rad (1 mentions)
7 protocols using cocktail protease inhibitors 1x
1
Protein Isolation from Cell Cultures
2
Mitochondrial Isolation from Cells
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The mitochondrial fraction was isolated using a mitochondria lysis buffer (10 mM Tris-HCl, 0.25 mM sucrose, 0.2 mM EDTA, pH 7.8), supplemented with protease inhibitors cocktail (1X) (Sigma-Aldrich; Merck KGaA). Lysed cells were centrifuged at 1,000 × g for 5 min at 4°C to pellet mitochondria and were deep frozen for 30 min at −70°C. This step was repeated three times. Defrosted samples were disrupted by repeatedly passages through a 21-gauge needle and centrifuged at 1,000 × g for 10 min at 4°C to pellet nuclei and cellular debris. Post-nuclear supernatant obtained was then centrifuged at 10,000 × g for 15 min at 4°C to collect intact mitochondria, which were resuspended in mitochondria storage buffer (Qproteome Mitochondria Isolation Kit; cat. no. 37612; Qiagen GmbH) for storage at −20°C and the analysis of mitochondrial proteins (SCO2 and FXN) by WB (54 (link)).
3
Molecular Profiling of Dystrophic Diaphragm Muscles
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Diaphragm muscles were isolated from 2-, 4-, and 24-week-old mdx and mdx/IL6 mice and immediately frozen in liquid nitrogen. Each sample (liquid nitrogen powdered diaphragm muscles) was homogenized in protein lysis buffer [Tris-HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, cocktail protease inhibitors/1X (Sigma Aldrich)]. Western blotting analysis was performed using 70 μg of protein extracts, and filters were blotted with antibodies against gp91phox (BD Transduction Laboratories), Nrf2 (Santa Cruz Biotechnology), NFκB p65 (ser536; Cell Signaling), NFκB (Cell Signaling), α-tubulin (Sigma Aldrich), β-tubulin (Cell Signaling), Glu-tubulin (detyrosinated α-tubulin; Abcam), and GAPDH (Santa Cruz Biotechnology). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
4
Protein Expression Analysis in ALS Model
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C2C12 and C2C12 MLC/SOD1G93A cells grown in 60 mm culture dishes were washed twice with cold phosphate-buffered saline, pelleted, resuspended in 100 uL of modified lysis buffer (Tris-HCl, ph 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, and cocktail protease inhibitors/1X (Sigma Aldrich)), and processed for western blot analysis. Filters were blotted with antibodies against hSOD1 (Santa Cruz), gp91phox (BD) anti-Perilipin 2 (Lifespan Biosciences), phospho-p42/p44 MAPK (Millipore), p42/p44 MAPK (Cell Signaling) phospho-Akt (Thr 308) (Sigma Aldrich), Akt (Cell Signaling), phospho-P70 (Thr389) (Cell Signaling), p70 (Cell Signaling), and HDAC4 (Cell Signaling). Protein levels of α-tubulin were used as control for equal protein loading. Signals were acquired by ChemiDoc-It Imaging System (UVP, LLC) and the analysis was performed using VisionWorks LS Image Acquisition analysis software.
5
Analyzing Dystrophic Diaphragm Muscle Signaling
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Diaphragm muscles from at least five animals/strain (wild-type, mdx and mdx/IL6 mice) were homogenized in modified lysis buffer [Tris–HCl, ph 7.5/20 mm , EDTA/2 mm , EGTA/2 mm , sucrose/250 mm , DTT/5 mm , Triton-X/0.1%, PMSF/1 mm , NaF/10 mm , SOV4/0.2 mm , cocktail protease inhibitors/1X (Sigma)]. Filters were blotted with antibodies against: Pax-7, Desmin, NFkBp65 (ser536), NFkB (Cell Signaling); GAPDH (Santa Cruz). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software. Elisa assay was performed to detect murine and transgenic IL-6 using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol.
6
Analyzing Muscle Protein Expression
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Diaphragm muscles from at least 3 animals/strain (wild type, mdx, and mdx/mIGF-1 mice) were homogenized in modified lysis buffer (Tris-HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, cocktail protease inhibitors/1X (Sigma)). Muscle lysates were processed as previously described (Pelosi et al., 2007 (link)). Filters were blotted with antibodies against: HDAC2 (Santa Cruz Biotechnology, INC), HADAC4 (Santa Cruz Biotechnology, INC) and αTubulin (Sigma). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis were performed with VisionWorks® LS Image Acquisition and Analysis Software (UVP, LLC).
7
Quantifying Oxidative Stress in Diaphragm Muscle
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Diaphragm muscles were isolated from 24-week-old wild-type and NSE/IL-6 mice, and liquid nitrogen powdered samples were homogenized in protein lysis buffer (Tris-HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, Sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV4/0.2 mM, Cocktail Protease Inhibitors/1x (Sigma-Aldrich)). For western blotting analysis, 70 μg of protein extract was used, and filters were blotted with primary antibodies against gp91phox (BD Transduction), G6PD (Santa Cruz), Nitrotyrosine (Millipore), and GAPDH (Santa Cruz) and appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories). Nitrotyrosine quantification was performed using the Stain-Free blot method for normalization (Criterion TGX Stain-Free Precast Gels; Bio-Rad).
Signals were captured by Chemi Doc™ XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
For the evaluation of circulating human IL-6, an ELISA was performed on serum samples using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to the manufacturer's protocol.
Signals were captured by Chemi Doc™ XRS 2015 (Bio-Rad Laboratories), and densitometric analysis was performed using Image Lab software (version 5.2.1; Bio-Rad Laboratories©).
For the evaluation of circulating human IL-6, an ELISA was performed on serum samples using Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to the manufacturer's protocol.
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