Introduction

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).

Specifications

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
Formula: C17H26N8O5-HCl
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).

Handling Blasticidin

  • Always wear gloves, mask, a laboratory coat, and safety glasses when handling Blasticidin containing solutions.
  • Weigh out Blasticidin and prepare solutions in a hood.

TOP

Preparing and Storing Stock Solutions

  • 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.

TOP

Blasticidin Selection in E. coli

For selection of Blasticidin-resistant E. coli, use Low Salt LB medium (10 g Tryptone, 5 g NaCl, 5 g Yeast Extract) containing 50-100 μg/ml Blasticidin. Depending on the bacterial strain used, optimize the Blasticidin concentration. If you get a lawn of bacteria on your Low Salt LB plate instead of individual bacterial colonies, increase the Blasticidin concentration to 100 μg/ml in the plate.

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.
TOP

Blasticidin Selection in Yeast

The concentration of Blasticidin required for selection in yeast varies depending on the species, strain, and type of medium used. Use 25-300 μg/ml Blasticidin for selection in yeast. We recommend performing a kill curve for each species, strain, and medium used to determine the appropriate Blasticidin concentration to use for selecting resistant cells.
TOP

Blasticidin Selection in Mammalian Cells

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

  1. Plate cells at approximately 25% confluence. Prepare a set of 6 plates. Allow cells to adhere overnight.

  2. The next day, substitute culture medium with medium containing varying concentrations of Blasticidin (e.g. 0, 2, 4, 6, 8, 10 μg/ml Blasticidin).

  3. Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.

  4. Determine the appropriate concentration of Blasticidin that kills the cells within 10-14 days after addition of the antibiotic.
TOP

Product Qualification

Blasticidin S HCl is lot qualified by performing a kill curve on Blasticidinsensitive and Blasticidin-resistant mammalian cell lines. Blasticidinsensitive cells should be killed at all concentrations tested (2.5-10 μg/ml) within 10 days after addition of Blasticidin.
TOP

Accessory Products

Media for bacteria and mammalian cells as well as transformation products (yeast and bacteria) and transfection reagents are available from Invitrogen. Visit www.invitrogen.com for details.

TOP

References

  1. 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.

  2. 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.

  3. 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.

  4. 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.
TOP
R210.pps     20-Feb-2007