• Packaged and labeled G418 disulfate.

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SKU: G001

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Description

G418 Disulfate (G418 sulfate) is routinely used for gene selection in cell culture.  G418 Disulfate is an aminoglycoside antibiotic isolated from Micromonospora rhodorangea and closely related to Gentamicin.

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    CAS Number

    108321-42-2

    Molecular Formula

    C20H40N4O10 · 2H2SO4

    Molecular Weight

    692.71

    Mechanism of Action

    G418 Disulfate, and other aminoglycosides prevent protein synthesis.  Resistance to G418 Disulfate is conferred by the neo gene (neomycin resistant gene) from either Tn5 or Tn601 (903) transposons. C ells 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,  which has negligible potency and has low-affinity for prokaryotic and eukaryotic ribosomes.

    Storage Conditions

    Ambient

    Tariff Code

    2941.90.1010

    Spectrum

    G418 Disulfate is toxic to susceptible bacteria and fungi.

Applications

    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.   TOKU-E scientists tested the selectivity factor of G418 for BHK-21 cells and HeLa cells.  Authors 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.

    Microbiology Applications

    G418 Disulfate is used as a gene selection agent during transfection of eukaryotic cells.

Specifications

    Form

    Powder

    Appearance

    White or off-white powder

    Source

    Micromonospora rhodorangea

    Biological Assay

    ED50 Resistant: ≥2500 ug/mL
    ED50 Sensitive: ≤400 ug/mL

    Elemental Analysis

    Carbon: 28.80-36.07%
    Hydrogen: 5.76-7.76%
    Nitrogen: 6.72-8.41%
    Water of Hydration: 0-6

    Ammonia

    ≤1.0%

    Water Content (Karl Fischer)

    ≤12.0%

    Potency (on a dry basis)

    ≥720 μg/mg (≥700 μg/mg As is)

    Absorbance

    1mg/mL:280nm <0.015
    100mg/mL: 570nm <0.10

    pH

    4.6-6.0 (200mg/mL)

    Optical Rotation

    +104º to +121º

References

    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 25:293-99. 

    Shin, Y (2007)  Selection of NptII transgenic wweetpotato plants Using G418 and paromomycin.  J. Plant Biol. 50(2):206-12

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