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Half-Life/Cycloheximide Chase Assay

RM Ritu Mishra
AB Akhil C. Banerjea
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Cycloheximide is a well-known translation inhibitor to analyze the half-life of cellular proteins. HEK-293T cells were used for cycloheximide chase assay in 6 well plate format with 1x105 cells per well. At ~70% confluency, cells were transfected with IRF9 only and IRF9 plus USP33 co-transfection in different plates with the help of Lipofectamine 2000. Post-24 hrs of transfection, Cycloheximide (#01810, Sigma-Aldrich) were given to all wells at 100µg/mL concentration. Cells were then harvested at various time points (2, 4, 8, 12 and 24 hours) followed by lysis with RIPA buffer and western blot analysis for indicated proteins.

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