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G418, Geneticin®
Commonly know as G418 or G-418, Geneticin® reagent is an aminoglycoside related to Gentamicin, and is commonly used as a selective agent for eukaryotic cells. As an analog of neomycin sulfate, it interferes with the function of 80S ribosomes and protein synthesis in eukaryotic cells.
The dominant-acting resistance genes (neor) used on common mammalian expression vectors contain elements derived from either transposons Tn601 (903) or Tn5. When grown in medium containing Geneticin® selection agent, stable colonies of mammalian cells expressing these resistance markers can be generated in 10 to 14 days.
What's in your G-418?
To be effective, selection agents need to be reliable, highly pure, and consistent in nature. Geneticin® selective antibiotic, considered the gold standard for nearly two decades, is the only G-418 reagent that delivers these qualities to your mammalian cell cultures.
Compared to other manufacturers’ G-418 products, Geneticin® antibiotic exhibits higher purity, so you can use less and produce greater selection pressure with minimal toxicity from contaminants.
When evaluating G-418 products, consider more than just potency. Look at purity and working range. Taken together, these three characteristics provide a true evaluation of the effectiveness of your G-418.
Purity. The purity of Geneticin® selection agent is consistently greater than 90%, as determined by HPLC — far higher than any other supplier. This means:
- 15-30% lower concentrations of Geneticin® agent can be used, as compared to other G-418 products, to achieve comparable selection results.
- Surviving clonal colonies may arise faster, and cells may appear healthier when using Geneticin® instead of lower purity G-418 products.
Potency. Potency as a descriptive term is often used synonymously with purity, but potency as reported in QC data is actually not related to purity. The potency value is a measure of bacterial growth inhibition.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
- Gentamicins that commonly contaminate G-418 preparations from other suppliers will add to potency in bacterial assays yet have no effect on mammalian cell selectivity.
- Other bacterial contaminants in impure G-418 may be toxic to mammalian cells, consequently lowering the ED 50 value.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
ED50 Assay. This is a true measure of eukaryotic growth selectivity as determined on reference NIH3T3 cells. Higher purity generally translates to higher ED50 values.
- Toxic impurities lower the ED50 value, which means a more narrow working range of antibiotic selection.
- A consistent ED50 from a supplier assures performance reproducibility from lot-to-lot in mammalian cell culture applications.
This means when purchasing a new lot of Geneticin®, you do not need to re-optimize for ideal antibiotic concentration (assuming that no other media alterations). The wide ranges of ED50 displayed in different lots from alternative G-418 suppliers may require you to re-optimize for each lot. Lot-to-lot consistency with Geneticin® is sufficiently high that you will not notice any difference in selection performance between lots.
Purity. The purity of Geneticin® selection agent is consistently greater than 90%, as determined by HPLC — far higher than any other supplier. This means:
- 15-30% lower concentrations of Geneticin® agent can be used, as compared to other G-418 products, to achieve comparable selection results.
- Surviving clonal colonies may arise faster, and cells may appear healthier when using Geneticin® instead of lower purity G-418 products.
Potency. Potency as a descriptive term is often used synonymously with purity, but potency as reported in QC data is actually not related to purity. The potency value is a measure of bacterial growth inhibition.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
- Gentamicins that commonly contaminate G-418 preparations from other suppliers will add to potency in bacterial assays yet have no effect on mammalian cell selectivity.
- Other bacterial contaminants in impure G-418 may be toxic to mammalian cells, consequently lowering the ED 50 value.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
ED50 Assay. This is a true measure of eukaryotic growth selectivity as determined on reference NIH3T3 cells. Higher purity generally translates to higher ED50 values.
- Toxic impurities lower the ED50 value, which means a more narrow working range of antibiotic selection.
- A consistent ED50 from a supplier assures performance reproducibility from lot-to-lot in mammalian cell culture applications.
This means when purchasing a new lot of Geneticin®, you do not need to re-optimize for ideal antibiotic concentration (assuming that no other media alterations). The wide ranges of ED50 displayed in different lots from alternative G-418 suppliers may require you to re-optimize for each lot. Lot-to-lot consistency with Geneticin® is sufficiently high that you will not notice any difference in selection performance between lots.
Table 1. Comparison of Geneticin® specifications with alternative G-418 suppliers.
| Specification | Invitrogen | Supplier A | Supplier B | The Geneticin® Advantage |
| Purity (HPLC) | >90-93% | 66-75% | 65-82% | Significantly higher purity |
| Potency claimed (µg/mg) | 718-735 | 712-724 | 673-735 | More stringent potency assay |
| Potency upon retest (µg/mg) | 718-735 | 640-659 | 621-677 | Consistently higher actual potency |
| ED50 Assay (µg/ml) | 2450-2700 | 1350-3100 | 600-2350 | Reliable selection lot-to-lot |
Table 1. Multiple lots of G-418 from two different suppliers were analyzed according to Invitrogen raw material QC protocols for Geneticin®. It was observed that results obtained with G-418 from Supplier A and Supplier B displayed wide variations on all measured parameters, including an inconsistency between the actual potency and the value reported on the supplied product literature. Discrepancies between re-tested potency values and that claimed by other suppliers may be due to differences in G-418 reference lots and assay procedures.
Figure 1. Geneticin® displays greater purity than G-418 from alternative suppliers.
Figure 1. Comparison of HPLC data of Geneticin® with selected lots of G-418 from Supplier A and Supplier B. Samples were prepared and analyzed according to the procedure published in Clinical Chem. (1978), Vol. 24, No.11, p1940.