Cells were lysed in lysis buffer A [20 mM HEPES (pH 7.5), 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, and protease cocktail I/II (Sigma-Aldrich)]. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were blocked in 5% skim milk in 0.01 M Tris-buffered saline (pH 7.5) containing 0.5% Tween 20 and then incubated with the appropriate primary antibodies. For cytochrome c release after treatment with SB743921 alone or in combination with bortezomib, subcellular fractions were prepared using the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, La Jolla, CA, USA). The purity of each fraction was verified using α-tubulin and peroxiredoxin 3 (Prx3) as specific markers of cytosol and mitochondria, respectively (16) (link).
Protease cocktail 1 2
Manufactured by Merck Group
Protease cocktail I/II is a laboratory reagent formulated to inhibit or inactivate a broad range of proteolytic enzymes. It is commonly used in protein extraction and purification protocols to prevent unwanted proteolysis. The product contains a mixture of protease inhibitors that target different classes of proteases, providing a versatile solution for sample preparation and downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using protease cocktail 1 2
1
Subcellular Protein Fractionation and Analysis
2
Western Blot Protein Analysis Protocol
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Cells were lysed in lysis buffer A [20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM EGTA, 1% Triton X-100, 10% glycerol, and protease cocktail I/II; Sigma], and cellular debris was removed by centrifugation at 10,000× g for 10 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, blocked with 5% skim milk in 0.01 M TBS (pH 7.5) containing 0.5% Tween 20, and blotted with the appropriate primary antibodies. The antigen–antibody complexes were detected by chemiluminescence (Abclone, Seoul, Korea). All of the uncropped images of western blot used in Figures can be found it Figure S9 .
3
Western Blot Protein Detection
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The cells were lysed in lysis buffer A (20 mM N-2-hydroxyethyl piperazine-Nʹ-2-ethanesulfonic acid [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2 mM ethylene glycol tetraacetic acid, 1% Triton X-100, 10% glycerol, and protease cocktail I/II; Sigma). Cell debris was removed by centrifugation at 10,000 × g for 10 min. The proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were then blocked with 5% skimmed milk in 0.01 M TBS (pH 7.5) containing 0.5% Tween 20 and blotted with the appropriate primary antibodies. The antigen–antibody complexes were detected using chemiluminescence (Abclone, Korea).
4
Western Blot Analysis of Protein Lysates
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Cells were lysed in lysis buffer A (20 mM N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2 mM ethylene glycol tetraacetic acid, 1% Triton X-100, 10% glycerol, and protease cocktail I/II; Sigma), and cellular debris was removed by centrifugation at 10,000 × g for 10 minutes. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, blocked with 5% skim milk in 0.01 M TBS (pH 7.5), containing 0.5% Tween 20, and blotted with the appropriate primary antibodies. The antigen-antibody complexes were detected by chemiluminescence (Abclone, Korea).
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