• Hygromycin B, EvoPure (Low Endotoxin)

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Description

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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    CAS Number

    31282-04-9

    Molecular Formula

    C20H37N3O13

    Molecular Weight

    527.52

    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 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.

    Tariff Code

    2941.90.1010

    Spectrum

    Hygromycin B is effective against eukaryotic and prokaryotic cells.

Applications

    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).

    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).

Specifications

    Form

    Powder

    Appearance

    White or off-white powder

    Water Content (Karl Fischer)

    Not more than 15.0%

    Melting Point

    160-180°C

    Sulfated Ash

    Reported on certificate of analysis

    Residual Solvents

    Reported on certificate of analysis

    Endotoxin

    Not more than 10 EU/mg

References

    References

    Dai S et al (2001)  Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol. Breeding. 7: 25–33

    Harrison S et al (2006)  A rapid and robust method of identifying tansformed Arabidopsis thaliana seedlings following floral dip transformation. Plant Methods 2(19):1-7  PMID 17087829

    González A, Jiménez A, Vázquez D, Davies JE, Schindler D. (1978)  Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.  Biochim Biophys Acta.  521(2):459-469  PMID 367435

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