Hygromycin B, EvoPure (Low Endotoxin)

- Overview
  - Hygromycin B, EvoPure® (Low Endotoxin) has a strict endotoxin content of ≤10 EU/mg as characterized by spectral data. The presence of endotoxin in Hygromycin B can decrease transfection efficiency and can even be toxic to resistant eukaryotic cells. This product may be suitable for use as an ancillary material for upstream pharmaceutical manufacturing.
    
    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.
    Mechanism of Resistance
    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
    
    White or off-white powder
    
    Solubility
    
    Freely soluble
    
    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: o Bacteria (E. coli) - 50 µg/mL - 100 µg/mL o Fungi - 100 µg/mL - 300 µg/mL o 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. 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 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).