G418 disulfate 50mg/ml packaged and labeled.

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  • Description

    G418 disulfate, also known as G418 sulfate, is routinely used as a selection antibiotic in cell culture gene selection applications. G418 disulfate is an aminoglycoside antibiotic isolated from Micromonospora rhodorangea and is closely related to the gentamicins; most notably, gentamicin B1. G418 is a generic name of Geneticin.®

    G418 disulfate 50 mg/mL (G021) is prepared as a solution at a concentration of 50 mg/mL in water for quick and easy dilution factor calculations.

     TOKU-E also offers G418 disulfate (G001) and G418 disulfate, EvoPure (G030) (≥99.0% pure).

    Mechanism of Action

    G418 disulfate, and other aminoglycosides, including kanamycin and neomycin, prevent protein synthesis by blocking the elongation step in prokaryotic and eukaryotic ribosomes.

    Mechanism of resistance:

    Resistance to G418 sulfate is conferred by the neo gene (neomycin resistant gene) from either Tn5 or Tn601 (903) transposons. Cells successfully transfected with resistance plasmids containing the neo resistance gene can express aminoglycoside 3'-phosphotransferase (APT 3' I or APT 3' II) which covalently modifies G418 to 3-phosphoric G418. 3-phosphoric G418 has negligible potency and has low-affinity for prokaryotic or eukaryotic ribosomes.

    Spectrum

    G418 disulfate is toxic to susceptible prokaryotic and eukaryotic cells including fungi (yeasts and molds), bacteria, mammalian and plant cells.

    • Microbiology Applications

      G418 disulfate can be used as a selection agent for G418 resistant bacteria or fungi after transformation.

    • Eukaryotic Cell Culture Applications

      G418 sulfate is routinely used as a selection agent in cell culture after transfection of eukaryotic cells. G418 resistant cells express the neogene which produces aminoglycoside 3'-phosphotransferase (APT 3' I or APT 3' II) - a protein that confers resistance to G418 sulfate and other aminoglycoside antibiotics.

      Optimal working concentration ranges of G418 sulfate are as follows:

      • Mammalian cell lines – 200 mg/L – 1000 mg/L
      • Bacteria and algae - ≤5 mg/L or less for bacteria and algae

      A working concentration of 200 mg/L is usually sufficient after resistant mammalian clones are selected and can be used for maintenance until stable resistant clones are selected.

      Note: The optimal working concentration of G418 sulfate to select for resistant mammalian clones depends on the cell lines used, the quality of G418 sulfate, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the neo resistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of G418 sulfate. A kill curve can be performed using the following concentrations of G418 sulfate:

      • 5 mg/L - 1400 mg/L for mammalian cells
      • 0.1 mg/L - 50 mg/L for bacteria (E. Coli ) and algae.

      High quality G418 sulfate ideally has high potency against sensitive cells (low ED50 concentration) and low potency against transfected resistant cells (high ED50 concentration). Impurities and certain bio active substances such as endotoxins present in G418 sulfate can be toxic to resistant cells and can cause death at relatively low concentrations.

      For more information on relevant cell lines, culture medium, and working concentrations, please visit the TOKU-E Cell-culture Database. 

      Suasnavas et al. used G418 disulfate from TOKU-E to select for resistant porcine TE and FF cells. Read more here: "Characterization and Potential Utility of Porcine Trophoblast-Derived Stem-Like Cells."

    • References

      Aragão F.J.L. and Brasileiro A.C.M., Positive, negative and marker-free strategies for transgenic plant selection. Braz. J. Plant Physiol., 14(1):1-10, 2002

      Davis, Bernard D. "Mechanism of Bactericidal Action of Aminoglycosides."Microbiological Reviews 51.3 (1987): 341-50.

      Dong Z.J. and McHughen, A. Improved procedure for production of transgenic flax plants using Agrobacterium tumefaciens. Plant Science, 88 (1993) 61-71 61. Elsevier Scientific Publishers.

    Form

    Solution

    Tariff Code

    2941.90.1010

    Related Documents


    For in vitro research use only. Not suitable for human or animal consumption. For MSDSs not available online, please email a request to [email protected]

  • CAS Number108321-42-2
    Molecular FormulaC20H40N4O10 · 2H2SO4
    Molecular Weight692.71 g/mol
    FormSolution
    AppearanceClear and colorless solution
    SourceMicromonospora rhodorangea
    Biological AssayED50 Resistant: Not less than 2,500µg/mL
    ED50 Sensitive: Not more than 400µg/mL
    Elemental AnalysisCarbon: 28.80 - 36.07%
    Hydrogen: 5.76 - 7.76%
    Nitrogen: 6.72 - 8.41%
    Waters of Hydration: 0 to 6
    AmmoniaNot more than 1.0%
    Water Content (Karl Fisher)(Powder) Not more than 12.0%
    Potency (on a dry basis)Not less than 720µg/mg
    Absorbance280nm (1mg/mL): Not more than 0.015
    570nm (100mg/mL): Not more than 0.10
    pH4.6 - 6.0
    Optical Rotation+104° to +121°
    Storage Conditions2-8°C
    Concentration50mg/mL
    IdentificationIR, HPLC - Passes test
  • Protocols

    G418 Disulfate Kill Curve Protocol


    Background:

    G418 disulfate, also known as G418 sulfate, is routinely used to select for successfully transfected mammalian cells that express the neo resistance gene in addition to the gene of interest. The neo gene encodes amino-glycoside 3’-phosphotransferase; an enzyme which confers resistance to G418 disulfate and neomycin. Before stable transfected cell lines can be selected, the optimal G418 disulfate concentration needs to be determined by performing a kill curve titration. The optimal concentration of G418 disulfate suitable for selection of resistant mammalian clones depends on the cell lines, media, growth conditions, and the quality of G418 disulfate. It is necessary to perform a kill curve for every new cell type and new batch of G418 disulfate.

    Preparation and storage of G418 disulfate solution:
    • Stock solution - dissolve G418 disulfate in water at a concentration of 50 mg/ml.
    • Sterile filter the solution using 0.45ilter.
    • Store solution at 2-8°C after use.


    Kill curve/G418 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, flow cytometry 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.
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    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.

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