Puromycin dihydrochloride is a protein synthesis inhibitor. Aminonuclease antibiotic used for selection and maintenance of cell lines expressing a transfected pac gene (S. alboniger), whose product, puromycin acetyltransferase, inactivates puromycin via acetylation; recommended concentration in cell culture 1-10µg/ml. Lot-to-lot variations in potency exist for all selection antibiotics, each new lot of puromycin should be titrated. The working puromycin concentration for mammalian cell lines ranges from 1-10 µg/ml. Prior to using the puromycin antibiotic, titrate the selection agent to determine the optimal concentration for target cell line. Use the lowest concentration that kills 100% of non-transfected cells in 3-5 days from the start of puromycin selection.
Puromycin Selection (shRNA protocol) for selection of stably transfected cells
- 48 hours post-transfection , aspirate the medium and replace with fresh medium containing puromycin at the appropriate concentration.
- Approximately every 2-3 days, aspirate and replace with freshly prepared selective media.
- Monitor the cells daily. Puromycin selection requires a minimum of 48 hours.
- Optimum effectiveness should be reached within 3-10 days.
- Assay transfected cells.
Suggested working conditions for selection in some mammalian cells:
- Hela human uterus: 3 µg/ml
- HEK293 human embryonic kidney: 3
- B16 mouse melanoma: 1-3
- PC1.0 hamster adenocarcinoma: 10
Titrating puromycin for selecting transfected cell lines
- Plate 2 x 105 cells in each well of a 6-well plate containing 3 ml of the appropriate complete medium plus increasing concentrations of puromycin (i.e., 0, 1.0, 2.5, 5.0, 7.5, and 10.0 µg/ml)
- Replace with fresh selective medium after 2 days to remove dead cells.
- Examine the wells for viable cells every two days.
- Monitor the cells daily and observe the percentage of surviving cells. Optimum effectiveness should be reached in 1-4 days.
- The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 3-5 days from the start of puromycin selection.