• Packaged and labeled G418 disulfate.

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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.

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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."

Wang, et al. used G418 disulfate from TOKU-E in RPMI media to select RFP expressing TC-2 cells. Read more here: "3D Porous Chitosan–Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer."

Baradaran-Heravi et al. used G418 disulfate from TOKU-E to study premature termination codon readthrough in H1299 cells. Read more here:"Novel small molecules potentiate premature termination codon readthrough by aminoglycosides."

    CAS Number

    108321-42-2

    Molecular Formula

    C20H40N4O10 · 2H2SO4

    Molecular Weight

    692.71

    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.

    Storage Conditions

    2-8°C

    Tariff Code

    2941.90.1010

    Spectrum

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

Applications

    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 neo gene 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.

    TOKU-E G418 stock solutions are prepared at 50mg/mL for quick and easy dilution factor calculations

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

    Microbiology Applications

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

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 Fisher)

    ≤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 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.

    Lin-Cereghino, Joan, et. al.. "Direct Selection of Pichia pastoris Expression Strains Using New G418 Resistance Vectors." Yeast 25 (2008): 293-99. Interscience.wiley.com. Web. 25 Apr. 2013.

    Shin, Young-Mi. "Selection of NptII Transgenic Sweetpotato Plants Using G418 and Paromomycin." Journal of Plant Biology 50.2 (2007): 206-12. Researchgate.net. Web. 25 Apr. 2013.

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