HEK-293T cells transfected with HA-AMPKα or FLAG-SENP2 were lysed in lysis buffer containing 50 mM Tris–Hcl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.5% Triton X-100, and protease inhibitors (Roche). After centrifugation, the lysates were incubated with anti-FLAG beads (SMART LIFESCIENCES) for 2 h. The beads were then washed four times with washing buffer (50 mM Tris–Hcl [pH 7.5], 100 mM NaCl, 5% glycerol, 0.1% Triton X-100, and protease inhibitors), and the immunoprecipitates were separated by SDS-PAGE for Western blotting.
Protease inhibitors
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Protease inhibitors are a class of laboratory equipment used to inhibit the activity of proteases, which are enzymes that break down proteins. They are commonly used in biological research and drug development to prevent the degradation of target proteins during experimental procedures.
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Lab products found in correlation
2 protocols using protease inhibitors
1
Immunoprecipitation of AMPK and SENP2
2
Detecting SUMOylated AMPKα in HEK-293T Cells
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The V5-SUMO plasmid, HA-AMPKα plasmid, FLAG-SENP2 plasmid, and FLAG-UBC9 plasmid were transfected into HEK-293T cells. HEK-293T cells were lysed in lysis buffer containing 50 mM Tris–Hcl (pH 8.0), 250 mM NaCl, 0.01% Tween, 1% Triton X-100, 0.0125 g/ml N-ethylmaleimide, and protease inhibitors (Roche). After centrifugation, lysates were incubated with anti-HA beads (SMART LIFESCIENCES) and rotated at 4 °C for 2 h. The beads were then washed four times with washing buffer (50 mM Tris–Hcl [pH 8.0], 250 mM NaCl, 0.01% Tween, 0.0125 g/ml N-ethylmaleimide, and protease inhibitors). Finally, the immunocoprecipitates were subjected to Western blotting using V5 antibody to detect SUMOylated AMPKα.
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