For whole cell extracts, HDFn cells were grown in 100-mm dishes to ~95% confluence. At the desired population doubling, cells were washed with cold 1X phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer [150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris-HCl (pH 8.0)] containing protease inhibitors (cOmplete EDTA-free Protease Inhibitor Cocktail Tablet; Sigma-Aldrich, St. Louis, MO, USA) for 2 min. The cells were scraped from the surface of the flasks, transferred to microcentrifuge tubes and centrifuged at 20,000 × g for 5 min. The soluble fractions were transferred to new tubes. For cellular fractionation, the cells were washed with cold 1X PBS and fractionated as previously described (32 (link)). Protein concentrations of all samples were determined using the Pierce® BCA Protein assay kit (Thermo Fisher Scientific, Inc., Chicago, IL, USA).
Complete edta free protease inhibitor cocktail tablet
Manufactured by Merck Group
Sourced in United States
The COmplete EDTA-free Protease Inhibitor Cocktail Tablet is a laboratory reagent designed to inhibit a wide range of proteases in protein samples. It is formulated without EDTA, making it suitable for applications where EDTA-sensitive downstream analyses are required.
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2 protocols using complete edta free protease inhibitor cocktail tablet
1
Whole Cell Extract Preparation for HDFn Cells
2
Immunoprecipitation of Synaptosomal Proteins
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For immunoprecipitation experiments, all steps were performed at 4°C in the presence of protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail tablet; Sigma-Aldrich) and phosphatase inhibitors (phosphatase inhibitor cocktail 2 and 3; Sigma-Aldrich). P3 (synaptosomal membrane fraction) was prepared as described above and resuspended in immunoprecipitation lysis buffer [50 mM tris-HCl, 150 mM NaCl, and 1% DMM (n-dodecyl β-d -maltoside; Sigma-Aldrich)]. Protein concentration was measured by BCA. P3 lysate (4 mg) was incubated with 3 μg of antibody or with an equivalent amount of IgG control for 1 hour on a rotating wheel before the addition of 30 μl of Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific) for an additional 3 hours. Following incubation, samples were washed four times with washing buffer [50 mM tris-HCl, 120 mM NaCl, and 0.5% DMM (Sigma-Aldrich)] and proteins were eluted with 1× Laemmli sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting.
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