Hygromycin B Solution (Low Endotoxin)

- Overview
  - Hygromycin B Solution (Low Endotoxin) is filter-sterilized, 50 mg/ml in PBS buffer. The presence of endotoxin in Hygromycin B can decrease transfection efficiency and can even be toxic to resistant eukaryotic cells.
    
    Hygromycin B is a unique aminoglycoside antibiotic derived from Streptomyces hygroscopicus routinely used as a selective agent in cell culture or microbiology applications to isolate Hygromycin B resistant cells after transfection or transformation, respectively.
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    Background
    
    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 of neomycin, gentamicin, and G418.
    
    Hygromycin B resistance is conferred by the hph gene isolated from Streptomyces hygroscopicus , 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.
- Properties
  - CAS Number
    
    31282-04-9
    
    Molecular Formula
    
    C20H37N3O13
    
    Molecular Weight
    
    527.52
    
    Appearance
    
    Clear and yellowish solution
    
    Other Properties
    
    Assay Hygromycin B ≥ 900 µg/mg
    Concentration: 50 +/- 10 mg/ml
    Purity Level: Hygromycin B: ≥ 90.0%
    
    Storage
    
    2-8°C
    
    * For research use only
- Applications
  - Application Description
    
    Spectrum: Hygromycin B is effective against eukaryotic and prokaryotic cells.
    
    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
    Yeast: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. A rapid method to screen for Hygromycin B resistant Arabidopsis in less than four days has been developed. After Arabidopsis seeds have been transformed with a resistance plasmid (pBIG-HYG), they are plated on MS medium with Hygromycin B and subjected to a two day stratification at 4°C in the dark. Seeds are then exposed to light for 4-6 hours to stimulate germination and then placed in the dark for another two days. Transformed seeds are selected and identified after a 24 hour period in the light. Resistant transformants are characterized by long hypocotyls. (Harrison et al, 2006).
    
    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.