Half-Life/Cycloheximide Chase Assay

For detail protocol, researchers may also kindly visit our previous paper, published in 'Blood' journal.

"Inhibition of β-TrcP–dependent ubiquitination of p53 by HIV-1 Vpu promotes p53–mediated apoptosis in human T cells 

Sachin Verma, Amjad Ali, Sakshi Arora, Akhil C. Banerjea

https://doi.org/10.1182/blood-2011-01-333427

1. Untransfected (1.2 × 10⁶) or transfected (2 μg of Wt or mutant ) HEK293T cells in a 35-mm dish were incubated for 36 hours. Your experiment and treatment could be anything related to your research question. Subsequently, 
2. cycloheximide (Sigma-Aldrich) (dissolve in DMSO) was added to a final concentration of 100 μg/mL, (stock solution of cycloheximide is prepared at 100 mg/ml in DMSO.
3. and cells were harvested after the indicated time points (It can vary depending on your protein and stability in question. Generally from 0 hr to 24 hrs time span is taken. We harvest treated cells at 0 h, 2 h, 4 h, 8h, 12h and 24 h. 
4. The lysate was prepared, and immunoblotting was performed as described in figure. and check the degrading trend of your gene of interest by applying specific antibody.