Western blot analysis was performed as previously described 31 (link). Briefly, MDSCs were lysed in Cell Lytic MT lysis buffer (Sigma) with Protease Inhibitor Cocktail (Invitrogen) and phosphatase inhibitor 2 and 3 (Sigma) for 15 min on a shaker. After centrifugation for 20 min at 12 000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad). Western blot analysis was performed using antibodies against mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, S6, and phospho-S6 (rabbit monoclonal antibodies, 1: 1 000, Cell Signaling). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1: 2 000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1: 2 000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce).
Cell lytic mt lysis buffer
Manufactured by Merck Group
Sourced in United Kingdom, United States
Cell Lytic MT lysis buffer is a solution used for the lysis of eukaryotic cells. It is designed to efficiently disrupt cell membranes and release cellular contents, including proteins and other biomolecules, for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Anti rabbit igg secondary antibodies conjugated with horseradish peroxidase, by Cell Signaling Technology (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Phosphatase inhibitor 2 and 3, by Merck Group (2 mentions)
Rabbit monoclonal anti β actin, by Cell Signaling Technology (2 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Protein a g agarose beads, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Sds page loading buffer, by Thermo Fisher Scientific (1 mentions)
Anti human fc, by Jackson ImmunoResearch (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Amido black stain, by Merck Group (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Protease and phosphatase inhibitor mix, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Phosphorylated akt ser473, by Cell Signaling Technology (1 mentions)
6 protocols using cell lytic mt lysis buffer
1
Western Blot Analysis of mTOR Pathway
2
Western Blot Analysis of mTOR Pathway
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Western blot analysis was performed as previously described 31 (link). Briefly, MDSCs were lysed in Cell Lytic MT lysis buffer (Sigma) with Protease Inhibitor Cocktail (Invitrogen) and phosphatase inhibitor 2 and 3 (Sigma) for 15 min on a shaker. After centrifugation for 20 min at 12 000×g (4°C), the supernatants were saved and protein concentrations of the samples were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 μg) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes (BioRad). Western blot analysis was performed using antibodies against mTOR, phospho-mTOR, p70S6K, phospho-p70S6K, S6, and phospho-S6 (rabbit monoclonal antibodies, 1: 1 000, Cell Signaling). Antibody against β-actin (rabbit monoclonal anti-β-actin, 1: 2 000, Cell Signaling) was used as a loading control. For detection, the membrane was incubated with anti-rabbit IgG secondary antibodies conjugated with horseradish peroxidase (1: 2 000, Cell Signaling). Bands were visualized using SuperSignal West Pico Chemiluminescent substrate (ThermoScientific Pierce).
3
Quantifying 35 K-Fc in Plasma
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35 K-Fc was detected in plasma by immunoprecipitation with Protein A/G agarose beads (Sigma, UK). Plasma samples were diluted 1:2 with Cell-lytic MT lysis buffer (Sigma, UK) and incubated with Protein A/G beads overnight at 4 °C with rotational mixing. The beads were washed 5 times with the lysis buffer prior to solubilisation of the bound proteins by LDS containing SDS-PAGE loading buffer (Invitrogen, UK). The resulting protein fraction was run on a western blot on a Nu-Page gel (Invitrogen) and transferred onto PVDF membrane (Amersham). The proteins were detected by blotting with anti-35 K antibodies (R&D Systems) and anti-human Fc (Jackson Labs, US) antibodies.
4
Western Blot Analysis of Sciatic Nerve Proteins
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Sciatic nerve segments were lysed and homogenised in Cell Lytic MT lysis buffer with protease inhibitor cocktail (Sigma-Aldrich). Protein samples (20 μg) were treated with reducing agent and sample buffer (Invitrogen) before 10 min incubation at 90 °C. Once cooled samples were run on SDS-PAGE gels (Invitrogen, NuPAGE 4–15% Bis-Tris gel) and transferred to nitrocellulose using an iblot (Invitrogen). The Western blot was performed using standard techniques, visualised and captured using the Azure C500 (Azure Biosystems) and densitometry analysis was performed using Fiji. Whole protein was used as a loading control with the amido black stain (Sigma) where densitometry analysis for each entire lane was used to normalise in Fiji, after being captured using the AzureC500 (Azure Biosystems)67 (link).
5
Western Blot Analysis of Protein Targets
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Western blot analysis was performed by SDS/PAGE as described previously [19 ]. Tissues and cells were lysed with Cell LyticMT lysis buffer (Sigma) which contained a protease and phosphatase inhibitor mix (Roche Molecular Biochemicals, Indianapolis, IN, USA). The lysis process lasted for 20 min on ice. The protein content of the samples was determined by BCA assay (Pierce, Rockford, IL, USA) after centrifugation for 15 min at 12,000 g (4 °C). Equal amounts of protein (30 μg) were loaded onto SDS/PAGE and blotted onto nitrocellulose membranes (Whatman, Clifton, NJ, USA). Immunobloting was performed with antibodies directed against each target molecule: Akt (1:1000; Cell signaling, Danvers, MA, USA); phosphorylated- Akt ser473 (1:500; Cell signaling, Danvers, MA, USA); eNOS (1:1000; Cell signaling, Danvers, MA, USA), phosphorylated-eNOS ser1177 (1:500; Cell signaling, Danvers, MA, USA), HMGB1 (1:1000; Abcam, Cambridge, MA, USA), VEGF (1:1000, Abcam, Cambridge, MA, USA), β-actin (1:10000; Cell signaling, Danvers, MA, USA), GAPDH (1:1000, Cell signaling, Danvers, MA, USA), α-Tubulin (1:1000; Cell signaling, Danvers, MA, USA). Proteins were detected by ECL chemiluminescence after incubation with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). NIH Image J software was used to quantify the bands.
6
Western Blot Analysis of Endothelial Cells
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