Puromycilation assay

Puromycin Assay for detection of newly synthesized proteins

1. Puromycin Dihydrochloride powder (Sigma-CAS 58-58-2) is dissolved in milli-Q water and stored at -20 °C in 1 ml aliquots (Stock concentration - 25 mg/ml).
2. Treat cell lines with puromycin (final concentration – 10 micrograms/ml) in media for 20 min. Wash cells with 1X PBS and collect them for lysis. Have “no puro treated” cells as a negative control for the assay (add same volume of water as vehicle).
3. Run Western blot using the lysates (loading 15-30 micrograms of protein/well) and probe the membrane with anti-Puromycin primary antibody (12D10, Millipore, MABE343) in 1:1000 concentration in 5% BSA/1X TBST at 4 °C overnight. 
4. Use anti-Mouse secondary antibody in desired concentration for 1 hr RT. 
5. Develop blot. Signal is usually detected within 3-4 min of exposure using regular ECL solution. Strip blot and probe the membrane with your preferred loading control (e.g. alpha-tubulin) (or take an image of the Ponceau Stained membrane to show equal loading).