Sciatic nerves of three different mice were pooled and flash frozen in liquid nitrogen immediately after isolation. Nerves were homogenized in a Precellys® 24 homogenizer (Bertin Instruments, Montigny‐le‐Bretonneux, France) in Pierce RIPA buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with cOmplete protease inhibitor and phosSTOP phosphatase inhibitor (Roche Diagnostics GmbH, Mannheim, Germany).
Complete protease inhibitor and phosstop phosphatase inhibitor
Manufactured by Roche
Sourced in Germany
Complete protease inhibitor and phosStop phosphatase inhibitor are lab equipment products that function to inhibit protease and phosphatase activity in samples. They are designed to preserve protein phosphorylation states and prevent unwanted proteolysis during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Modified bradford assay, by Bio-Rad (1 mentions)
Anti puromycin, by Developmental Studies Hybridoma Bank (1 mentions)
β actin antibody, by Merck Group (1 mentions)
4 protocols using complete protease inhibitor and phosstop phosphatase inhibitor
1
Extraction and Homogenization of Murine Sciatic Nerves
2
ELISA and Western Blot Analysis of Cell Apoptosis
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For ELISA and western blot analyses, cells were seeded on cell culture dishes (15 cm diameter) and grown for one to two days until they reached 80 to 85 % confluence. Full medium was replaced with medium containing 0.1 % BSA and cells were maintained in this medium for one hour before adding 0.1 μM GX15-070. For controls, Jurkat cells were centrifugated, resuspended in medium containing 0.1 % BSA and treated with staurosporine or a combination of staurosporine and Q-VD-OPh. After the treatment times indicated, medium was removed and cells were washed with ice-cold PBS. All further steps were performed on ice. Cell lysis was conducted in lysis buffer containing protease and phosphatase inhibitors (Complete protease inhibitor and phosStop phosphatase inhibitor, Roche Applied Science, Mannheim, Germany). The vehicle-stimulated control cells were maintained for 24 h in medium containing BSA before lysis. Lysates were clarified by centrifugation at 10,000 × g for 10 min at 4 °C. Protein concentration was determined with a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).
3
ELISA and Western Blot Analyses of Cell Signaling
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For the ELISA and western blot analyses, cells were seeded on cell culture dishes (15 cm diameter) and grown for 1–2 days until they reached 80–85 % confluence. Full medium was replaced with medium containing 0.1 % BSA and cells were maintained in this medium for 1 h before 3 µM 7BIO or vehicle was added. Non-adherent Jurkat cells were harvested by centrifugation, resuspended in medium with 0.1 % BSA and staurosporine or a combination of staurosporine and Q-VD-OPh was added, respectively. After the indicated stimulation times, the medium was removed and the cells were washed with ice-cold PBS. All additional steps were performed on ice. For the cell lysis, a lysis buffer containing protease and phosphatase inhibitors (Complete protease inhibitor and phosStop phosphatase inhibitor, Roche Applied Science, Mannheim, Germany) was used. The lysates were clarified by centrifugation at 10,000×g for 10 min at 4 °C. The protein concentration was determined with a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).
4
Puromycin-Based Protein Synthesis Assay
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Cells were plated as described above in 35 mm dishes. After 12 h of culture in treatment medium, 10 µM puromycin was added to the medium, and cells were harvested 10 min after the addition of puromycin. Cells were washed twice with ice-cold PBS and were then lysed in 100 μL of cell lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% v/v Nonidet NP-40) supplemented with Complete protease inhibitor and PhosSTOP phosphatase inhibitor (Roche). Cell lysate was mixed with an equal volume of 2X SDS PAGE loading buffer (0.1 M Tris-HCl, pH 6.8, 4% w/v SDS, 20% v/v glycerol, 200 mM dithiothreitol, and 0.05% w/v bromophenol blue). Proteins were separated by SDS-PAGE (10% w/v polyacrylamide) and were then transferred to Immobilon-P membranes. Membranes were blocked for 1 h in Tris buffered saline (50 mM Tris, 150 mM NaCl, pH 7.6) containing 5% w/v nonfat milk and 0.1% v/v Tween-20. Puromycin-labeled polypeptides were then quantified by incubating membranes with anti-puromycin (Developmental Studies Hybridoma Bank, #PMY-2A4) overnight at 4 °C and then with horseradish peroxidase-coupled secondary antibodies at room temperature for 1 h. Immunoblots were visualized using enhanced chemiluminescence and density was measured using ImageJ software. β-Actin antibody (Sigma-Aldrich) was used to quantify β-actin as a loading control.
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