SKU: G030  / 
    CAS Number: 108321-42-2

    G418 Disulfate, EvoPure®

    $115.00 - $311.00
    G418 Disulfate, EvoPure®

    G418 Disulfate, EvoPure® is a highly pure (≥99.0%) version of G418 Disulfate. It has been purified to remove the impurities commonly present in G418 Disulfate. This aminoglycoside antibiotic, originally isolated from Micromonospora rhodorangea, is routinely used for gene selection in cell culture.

    We also offer:

    • G418 Disulfate ReadyMadeTM Solution (G020-G021) 
    • G418 Disulfate (G001)
    • G418 Disulfate (Low Endotoxin)(G048)
    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 Disulfate 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 bacteria and fungi
    Microbiology Applications G418 Disulfate is used as a gene selection agent during transfection of eukaryotic cells.
    Eukaryotic Cell Culture Applications G418 Disulfate is routinely used as a selection agent in cell culture after transfection of eukaryotic cells.   Resistant cells express the neo gene which produces aminoglycoside 3'-phosphotransferase (APT 3' I or APT 3' II), a protein that confers resistance to G418 Disulfate and other aminoglycoside antibiotics.

    Optimal working concentrations:

        • Mammalian cell lines:  200 mg/L – 1000 mg/L
        • Bacteria and algae:  ≤5 mg/L

    The optimal working concentration of G418 Sulfate to select for resistant clones depends on the cell line, reagent quality, reagent lot, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and plasmid quality.  A kill curve should  be performed to determine the optimal  concentration for each experimental system.

    Use the following guide to determine the concentration to use to generate a kill curve:

        • 5 mg/L - 1400 mg/L (mammalian cells)
        • 0.1 mg/L - 50 mg/L (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.

    The Selectivity Factor is a quantifiable measure of how efficient an antibiotic is during the process of gene selection.   Our technical team tested the selectivity factor of G418 for BHK-21 cells and HeLa cells.  We found that G418 is an ideal selection antibiotic for transfected BHK-21 cells but not optimal for HeLa cells.  The method uses a modified MTT assay, which can be used to numerically determine the antibiotic efficiency (Delrue I et al, 2018).   For more information about the Selectivity Factor, click here.

    For more information on relevant cell lines, culture medium, and working concentrations, please visit the TOKU-E Cell-culture Database.
    Molecular Formula C20H40N4O10 · xH2SO4 (lot specific)
    Source Biosynthetic: produced by Micromonospora rhodorangea.
    Absorbance 1mg/ml (water): 280nm <0.015 570nm <0.01 100mg/ml (water): 570nm <0.01 1.74g/25 ml (water): 280nm <0.7
    Documents G418 Protocol.pdf|Plasmid_DNA_Transfection_Protocol.pdf|Selection_of_Stable_Transfected_Cell_Lines_Protocol.pdf
    References

    Aragão FJL and Brasileiro ACM (2002)  Positive, negative and marker-free strategies for transgenic plant selection. Braz. J. Plant Physiol. 14(1):1-10

    Davis BD (1987)  Mechanism of bactericidal action of aminoglycosides.  Microbiol. Rev. 51(3):341-50

    Delrue I, Pan Q, Baczmanska AK, Callens BW and Verdoodt LLM (2018)   Determination of the selection capacity of antibiotics for gene selection.   Biotechnol. J. 13(8):1700747  PMID 29436782

    Lin-Cereghino J et al (2008)  Direct selection of Pichia pastoris expression strains using new G418 resistance vectors.  Yeast 25:293-299

    Shin Y (2007)  Selection of NptII transgenic sweet potato plants Using G418 and paromomycin.  J. Plant Biol. 50(2):206-212
    Protocols

    G418 Disulfate Kill Curve Protocol


    Background:

    G418 Disulfate (syn: G418 Sulfate; Geneticin) 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 0.4 5 µm filter.
    • 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/well) 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.
    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.
    MIC Enterobacter| 1 － >64| Escherichia coli (animal health isolate)| 1 － >64| Klebsiella| 1 － 32|