Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two Blasticidin S deaminase genes: BSD from Aspergillus terreus (Kimura e al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert Blasticidin S to a nontoxic deaminohydroxy derivative (Izumi et al., 1991).
Contents: 50 mg supplied as white, solid powder.
Shipping/Storage: Shipped at room temperature. Store at -20°C.
Merck Index: 12: 1350
Molecular Weight: 458.9
E. coli Selection: 50-100 μg/ml in low salt LB medium*
*(NaCl concentration should not exceed 5 g/liter).
Yeast Selection: 25-300 μg/ml in suitable medium
Mammalian Cells 2-10 μg/ml in suitable medium (varies with cell Selection: line).
- Always wear gloves, mask, a laboratory coat, and safety glasses when handling Blasticidin containing solutions.
- Weigh out Blasticidin and prepare solutions in a hood.
- Prepare Blasticidin stock solutions of 5-10 mg/ml by dissolving Blasticidin in sterile water and filter-sterilize the solution. Blasticidin is soluble in water and acetic acid.
- Aliquot in small volumes suitable for one time use and store at 4ºC (short term) or at -20ºC (long-term). Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).
- Aqueous stock solutions are stable for 1-2 weeks at 4ºC and 6-8 weeks at -20ºC. Medium containing Blasticidin may be stored at 4ºC for up to 2 weeks.
- pH of the aqueous solution should not exceed 7.0 to prevent inactivation of Blasticidin.
- Upon thawing, use what you need and discard the unused portion.
Note that the salt concentration of the medium must remain low (<90 mM) and the pH should not exceed 7.0. Failure to lower the salt content of your LB medium results in nonselection due to Blasticidin inhibition unless a higher Blasticidin concentration is used.
The Blasticidin concentration required for selection in mammalian cells varies depending on the cell line used. Use 2-10 μg/ml Blasticidin for selection in mammalian cells. We recommend performing a kill curve as describe below to determine the appropriate Blasticidin concentration to use for selecting resistant cells.
Determining Blasticidin Sensitivity
- Plate cells at approximately 25% confluence. Prepare a set of 6 plates. Allow cells to adhere overnight.
- The next day, substitute culture medium with medium containing varying concentrations of Blasticidin (e.g. 0, 2, 4, 6, 8, 10 μg/ml Blasticidin).
- Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.
- Determine the appropriate concentration of Blasticidin that kills the cells within 10-14 days after addition of the antibiotic.
- Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., and Hanaoka, F. (1991). Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells. Exp. Cell Res. 197, 229-233.
- Kimura, M., Takatsuki, A., and Yamaguchi, I. (1994). Blasticidin S Deaminase Gene from Aspergillus terreus (BSD): A New Drug Resistance Gene for Transfection of Mammalian Cells. Biochim. Biophys. ACTA 1219, 653-659.
- Takeuchi, S., Hirayama, K., Ueda, K., Sakai, H., and Yonehara, H. (1958). Blasticidin S, A New Antibiotic. The Journal of Antibiotics, Series A 11, 1-5.
- Yamaguchi, H., Yamamoto, C., and Tanaka, N. (1965). Inhibition of Protein Synthesis by Blasticidin S. I. Studies with Cell-free Systems from Bacterial and Mammalian Cells. J. Biochem (Tokyo) 57, 667-677.