• Hygromycin B packaged and labeled.

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SKU: H007

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Hygromycin B is a unique aminoglycoside antibiotic derived from Streptomyces hygroscopicus. Hygromycin B is routinely used as a selective agent in cell culture or microbiology applications to isolate hygromycin B resistant cells. TOKU-E recommends preparing a stock solution at 50 mg/mL - 100 mg/mL. TOKU-E also offers hygromycin B solution conveniently prepared at 50 mg/mL in PBS buffer.

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    Mechanism of Action

    Hygromycin B inhibits protein synthesis by strengthening the interaction of tRNA binding in the ribosomal A-site. Hygromycin B also prevents mRNA and tRNA translocation by an unknown mechanism. These are unique mechanisms for an aminoglycoside antibiotic and they differ from the mode of action neomycin, gentamicin, and G418.

    Mechanism of resistance:

    Hygromycin B resistance is conferred by the hph gene and is isolated from Streptomyces hygroscopicus DNA. The hph hygromycin B resistance gene sequence is a 1467 bp fragment which encodes hygromycin B phosphotransferase (HPh). Cell lines successfully transfected with the hph gene produce hygromycin B phosphotransferase and convert hygromycin B to 7”-O-phosphoryl-hygromycin B by phosphorylating the 4-hydroxyl group on the cyclitol ring of hygromycin B. 7”-O-phosphoryl-hygromycin B lacks antibiotic activity and does not interact with prokaryotic or eukaryotic ribosomes.

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    Hygromycin B is effective against eukaryotic (mammalian) and prokaryotic (bacteria, fungi/yeast) cells.


    Eukaryotic Cell Culture Applications

    Hygromycin B is routinely used as a selective agent in mammalian cell culture to isolate hygromycin B resistant cells after transfection. Selectable markers for hygromycin B resistant cells include the hyg or hph resistance genes which express a phosphotransferase that inactivates hygromycin B by phosphorylation. Effective working concentrations range from 100 – 1000 µg/mL. The optimal working concentration of hygromycin B for selection of resistant mammalian clones depends on the cell lines used, hygromycin B quality, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the hph resistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of hygromycin B. Optimal selection concentrations of hygromycin B can range from 50 µg/mL - 1000 µg/mL; however, most common selection concentration ranges are between 50 µg/mL - 200 µg/mL.

    For additional information regarding relevant cell lines, resistance plasmids, and culture media, please visit our cell culture database.

    Microbiology Applications

    Hygromycin B can be used as a selection agent to isolate hygromycin b resistant bacteria and fungi. The following hygromycin B selection concentrations should serve as a guide only and may vary depending on experimental conditions and cells used:

  • Bacteria (E. coli) - 50 µg/mL - 100 µg/mL
  • Fungi - 100 µg/mL - 300 µg/mL
  • Yeasts - 50 µg/mL - 200 µg/mL
  • Plant Biology Applications

    Hygromycin B is routinely used as a selection agent for Arabidopsis plants that have been transformed with a hygromycin B resistance gene via Arabidopsis mediated transformation. A rapid method to screen for hygromycin B resistant Arabidopsis in less than four days has been developed and described by Samuel J Harrison et al. After Arabidopsis seeds have been transformed with a hygromycin B resistance plasmid (ex. pBIG-HYG), they are plated on MS medium with hygromycin B and subjected to a two day stratification period at 4°C in the dark. After stratification, seeds are exposed to light for 4-6 hours to stimulate germination and then placed in the dark for another two days. Transformed seeds are then selected and identified after a 24 hour period in the light. Hygromycin B resistant transformants are characterized by long hypocotyls (0.8 – 1.0cm vs. 0.2-0.4 cm).





    Off-White or Light Tan Powder


    Streptomyces Hygroscopicus.

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    ≥900 u/mg

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    Loss on Drying




    Dai S., Zheng P., Marmey P., Zhang S., Tian W., Chen S., Beachy R.N. and Fauquet C. Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Molecular Breeding 7: 25–33, 2001. © 2001 Kluwer Academic Publishers.

    Schindler, D. "Studies on the Mode of Action of Hygromycin B, an Inhibitor of Translocation in Eukaryotes." Nucleic Acids and Protein Synthesis 521.2 (1978): 459-69. www.ncbi.gov. Web. 6 Sept. 2012.

    Borovinskaya, Mari A. et al. "Structural Basis for Hygromycin B Inhibition of Protein Biosynthesis." Ncbi.nlm.nih.gov. N.p., n.d. Web. 5 Sept. 2014.

    Harrison, Samuel J. et al. "A Rapid and Robust Method of Identifying Transformed Arabidopsis Thaliana Seedlings following Floral Dip Transformation." Plant Methods 2.19 (2006): 1-7. Web. 4 Nov. 2014.

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