SKU: H024  / 
    CAS Number: 31282-04-9

    Hygromycin B, EvoPure® (Low Endotoxin)

    $332.00 - $537.00
    Hygromycin B, EvoPure® (Low Endotoxin)

    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 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.

    This product is considered a dangerous good. Quantities above 1 g may be subject to additional shipping fees. Please contact us for questions.

    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.
    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).
    Molecular Formula C20H37N3O13
    Identification
    • NMR: Confirmed
    • IR: Confirmed
    • MS: Confirmed 
    Documents Hygromycin Protocol.pdf|Plasmid_DNA_Transfection_Protocol.pdf|Selection_of_Stable_Transfected_Cell_Lines_Protocol.pdf
    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
    Protocols

    Hygromycin B Kill Curve Protocol


    Background:

    Hygromycin B is a unique aminoglycoside antibiotic produced by Streptomyces hygroscopicus and is routinely used as a selective agent in cell culture and microbiology applications to isolate transfected, hygromycin B resistant cells. Before stable transfected cell lines can be selected, the optimal hygromycin B concentration needs to be determined by performing a kill curve titration. The optimal working concentration of hygromycin B suitable for selection of resistant mammalian clones depends on the cell lines, media, growth conditions, and the quality of hygromycin B. Because of these variables, it is necessary to perform a kill curve for every new cell type and new batch of hygromycin B.

    Preparation and storage of hygromycin B solution:

    Hygromycin B is soluble in water at >50 mg/mL. It is also soluble in methanol or ethanol. Solutions should be sterilized by filtration, not by autoclaving.

    Hygromycin B solutions have been reported to lose activity on freezing. Since solutions are stable refrigerated, freezing should be avoided. Hygromycin B products should be stored as supplied at 2-8 °C. The dry solid is stable for at least five years if stored at 2-8 °C. Hygromycin B solutions are stable as supplied for two years if stored at 2-8 °C.

    Kill Curve/Hygromycin B Titration Protocol:

    1. Seed cells of the parental cell line in a 24-well plate at different densities (50,000 – 100,000 and 200,000 cells/ml) and incubate the cells for 24 hours at 37°C.
    2. Remove medium and then add medium with varying concentrations of antibiotic (0, 50, 100, 200, 400, 600, 800, and 1,000 μg/ml) and incubate at 37°C.
    3. Refresh the selective medium every 3-4 days and observe the percentage of surviving cells over time (e.g. by EMA vs Hoechst staining or MTT assay).
    4. Determine the lowest concentration of antibiotic that kills a large majority of the cells within 14 days. This concentration should be used for selection of a stable transfected cell line.
    5. If necessary, repeat the experiment to narrow the antibiotic concentration range.

     

    Plasmid DNA Transfection Protocol


    Background: 

    Once the appropriate antibiotic concentration to use for selection of the stable transfected cells has been determined by performing a kill curve, the next step is to generate a stable cell line by transfection of the parental cell line with a plasmid containing the gene of interest and an antibiotic resistance gene.

     

    Plasmid DNA Transfection Protocol:

    1. Seed the parental cell line in 24-well plate and incubate for 24h at 37°C.
    2. Transfect the parental cell line the next day at 80% confluency with the construct (e.g. using calcium phosphate etc…) and include a sample of untransfected cells as a negative control. Incubate at 37°C in C02.
    3. After transfection (6h to 24h depending on the transfection method used), wash the cells once with 1X PBS and add fresh medium containing the selection antibiotic to the cells. Use the appropriate antibiotic concentration as determined from the kill curve.
    4. Check, refresh, and expand the cells in selection medium every 2-3 days until you have enough cells for limited dilution (confluency in T25 flask or 10 cm dish).

    QC

    Seed 24-wells with insert and determine the transfection efficiency by immunostaining:

    1. Grow cells on insert in a 24-well plate until well is confluent.
    2. Remove medium and wash cells with 1X PBS.
    3. Fix cells with methanol or paraformaldehyde and wash with 1X PBS.
    4. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody).
    5. Wash cells with 1X PBS.
    6. Add secondary antibody in 24-well and incubate at 37°C for 1 hour depending on antibody).
    7. Wash with 1X PBS.
    8. Remove insert from 24-well plate and affix to microscopy slide with nail polish or other suitable adhesive.
    9. Determine the percentage of transfected cells with fluorescence microscope.
    Click here for PDF


    Selection of Stable Transfected Cell Lines Protocol

    Background:

    Once the cells have been successfully transfected, the next step is to seed and select the transfected cell line in a single 96-well plate to select pure colonies by limited dilution as outlined below:

     

    Protocol:

    1. Seed the transfected cells in 96-well plates in 10% conditioned medium
      • 2x96 well plate with 0.1 cell per well
      • 2x96 well plate with 0.5 cell per well
      • 2x96 well plate with 1 cell per well
    2. Incubate the cells for 24h.
    3. Remove medium and add conditioned selection medium containing selection antibiotic at the pre-determined concentration required for your cell line. Incubate 96-well plates at 37°C with C02.
    4. Check the plates every day for colonies. Colony formation depends on proliferation rate of the cell line and can take anywhere from 3 days to 1 week.
    5. Refresh selective medium every 3-4 days until colonies appear.
    6. Select the wells with only one single colony. Make sure colonies are not growing in clumps as they will be less sensitive to the antibiotic.
    7. When a well contains a single colony, transfer the colony to a 24-well in selection medium and so on until you have enough cells for freezing and storage in liquid nitrogen. Use the appropriate antibiotic concentration as determined from the kill curve.

     

    QC

    Seed 24-wells with insert for an immunostaining to determine percentage of cells expressing the gene of interest to be able to identify a 100% pure clone. You can also use Western blotting, flow cytometry or another technique depending on the cell line used.

    Seed 24-wells with insert and determine the expression level of the gene of interest by immunostaining:

    1. Grow cells on insert in a 24-well plate until well has confluent growth.
    2. Remove medium and wash cells with 1X PBS.
    3. Fix cell with methanol or paraformaldehyde and wash with 1X PBS.
    4. Add primary antibody in 24-well against protein of interest and incubate at 37°C for 1 hour (depending on antibody).
    5. Wash cells with 1X PBS.
    6. Add secondary antibody in 24-well plate and incubate at 37°C for 1 hour (time depends on antibody type).
    7. Wash cells with 1X PBS.
    8. Remove insert from 24-well plate and affix to microscopy slide with nail polish or other appropriate adhesive.
    9. Determine the percentage of transfected cells with fluorescence microscope.