G418 Disulfate (Low Endotoxin)

Produkt przeznaczony wyłącznie do badań. Nie nadaje się do spożycia. Więcej informacji na stronie producenta www.toku-e.com


G418 Disulfate (Low Endotoxin) has a strict endotoxin content of ≤ 1 EU/mg.  G418 Disulfate, an aminoglycoside antibiotic originally isolated from Micromonospora rhodorangea, is routinely used for gene selection in cell culture.  The presence of endotoxin in G418 Disulfate can decrease transfection efficiency and even be toxic to resistant eukaryotic cells.  

We also offer:

    G418 Disulfate (G001)
    G418 Disulfate Solution (50 mg/ml) in Water (G020-G021)
    G418 Disulfate, EvoPure (G030)

CAS Number: 108321-42-2
    
Molecular Formula: C₂₀H₄₀N₄O₁₀ · 2 H₂SO₄
    
Molecular Weight: 692.71
    
Mechanism of Action
G418 Disulfate, along with 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. 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, which has negligible potency and has low-affinity for prokaryotic and eukaryotic ribosomes.

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 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.

High quality G418 Disulfate has high potency against sensitive cells (low ED50 concentration) and low potency against transfected resistant cells (high ED50 concentration).   Impurities and certain bioactive substances such as endotoxins can be toxic.

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.

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 sweet potato plants Using G418 and paromomycin.  J. Plant Biol. 50(2):206-12