Cells in six-well plates were lysed with NP-40 buffer (Beyotime, Shanghai, China) with 0.1% mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor cocktail (Roche Molecular Biochemicals) for 30 min in ice. SDS was introduced to the supernatant and boiled for 10 min after centrifugation at 12,000 ×g for 10 min at 4°C. Before Western blotting, protein concentrations were determined by BCA assay. Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose filter (NC) membranes (Millipore, Billerica, MA), then blocked with 5% nonfat dry milk in TBST (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 2 h at room temperature, and then incubated with anti-EMP1 (ab230445, Abcam, Cambridge, MA) antibody or anti-GAPDH (LF205, Epizyme, Shanghai, China) antibody at 4°C overnight. After three rounds of washing with TBST, the membrane was incubated with a secondary antibody in PBS for 1 h at room temperature in the dark. After using TBST for washing three times, the membrane was scanned and analyzed in an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, Nebraska USA).
Pmsf protease inhibitor cocktail
Manufactured by Roche
Sourced in China
PMSF (Phenylmethylsulfonyl fluoride) is a protease inhibitor cocktail. It is used to inhibit the activity of various proteases in biological samples, such as serine proteases, cysteine proteases, and certain metalloproteases. PMSF is commonly used in protein purification, cell lysis, and other applications where the preservation of protein integrity is important.
Automatically generated - may contain errors
2 protocols using pmsf protease inhibitor cocktail
1
Western Blot Analysis of EMP1 Protein
2
TGEV Infection and Protein Analysis
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ST cells were infected with TGEV or transfected with plasmids in 6-well plates; each well of a 6-well plate was lysed with 100 μL of NP-40 buffer (Beyotime, China, Shanghai) containing 0.1% mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor cocktail (Roche Molecular Biochemicals) for 30 min in ice. After centrifugation for 12,000 × g for 10 min at 4°C, SDS was added to the supernatant and boiled for 10 min. Cell lysates were electrophoresed on SDS-PAGE gels, transferred to nitrocellulose filter (NC) membranes (Millipore, Billerica, MA, USA), then blocked with 5% nonfat dry milk in TBST (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Tween 20) for 2 h at room temperature, and incubated with the primary antibodies at 4°C overnight. After three washings with TBST, the membrane was incubated with the corresponding IRDye800-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) in PBS for 1 h at room temperature. After three washings with TBST, the membrane was scanned and analyzed in an Odyssey infrared imaging system (Li-Cor Biosciences).
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