G-418 FAQS (Frequently Asked Questions)

For the last 15 years, A.G. Scientific has been a leading manufacturer, and supplier of g-418, Geneticin®.  Our success with a combination of fermentation & synthesis that has allowed us to build a catalog of over 160 antibiotics and a customer base of researchers, catalog biochemical distributors and cell media manufacturers worldwide. Click here for our Comprehensive Antibiotic E-Guide. We offer the full range of services: custom bottling, sterile formulations, custom packaging, as well as, a full suite of private labeling capabilities.

The popular selection antibiotic, G-418, is a product we often get questions on solution storage & what are the procedures to determine toxic concentrations.  Here are the most Frequently Asked Questions (FAQS) we have received from our customers and answers. A Material and Safety (MSDS) document link is also available for download.

  • QUESTION: G-418 works on what cell types?
  • ANSWER: G-418 inhibits prokaryotic and eukaryotic cell's protein synthesis.  G-418 is toxic to bacteria, yeast, protozoans, higher plant, and mammalian cells.
  • QUESTION: What is the working concentration for selection?
  • ANSWER: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 10-2000 mg/ml. Commonly used concentrations for selection are 100-2000 mg/ml for mammalian cells, 10-100 mg/ml for plant cells, 500-1000 mg/ml for yeast, and 10-100 mg/ml for Dictyostelium amoebae. Your optimum concentration should be tested experimentally.
  • QUESTION: Is G-418 the same as Geneticin®?
  • ANSWER: G-418 and Geneticin are the same chemical structure. G-418 is the generic chemical while Geneticin® is a registered trademark brand of LifeTech Corporation.
  • QUESTION: What is the potency specification for G418 liquid and powder??
  • ANSWER:The potency specification is > 700 µg/mg. Small lot-to-lot variances do occur.
  • QUESTION: What gene confers resistance to G-418?
  • ANSWER: The neo / kan gene. This gene allows production of aminoglycoside 3-phosphotransferase, which inactivates G418, Neomycin and Kanamycin by phosphorylation.  QUESTION: How can non-transfected cells escape antibiotic selection? ANSWER: Cells can escape selection if the antibiotic is used at too low concentration or if the cell density on the plate is too high. Additionally, cells rapidly proliferating are killed faster than these, only slowly proliferating. Control cells should die within 5-7 days after addition of the antibiotic allowing colonies of resistant cells to form by 1014 days. G418 Chemical Structure
  • QUESTION: Can mammalian cells in culture divide in the presence of G418? ANSWER: Yes. Cells can continue to divide up to two times in the presence of lethal doses of G418. The effect of G418 usually becomes apparent by two days.
  • QUESTION: How do I work with the reported potency of G418?
  • ANSWER: The potency reported is microbiological potency. It is a function of G418 inhibitory effect on B. subtilis ATCC6633. Potency is reported in µg/mg. To convert the potency reported on the powder to liquid potency divide the potency by 1000 and multiply this by the desired potency. For example: If the potency reported on the powder is 750 µg/mg and you wish to make a solution of 400 mg/mL you would: 1000/750 x 400 mg/mL = 533.33 mg/mL We recommend that a dose response curve be set for both sensitive and resistant mammalian cells. Refer to the Certificate of Analysis under ED50 Sensitive and ED50 Resistant for suitable concentrations of G418 to use for the curve. These concentrations are reported as dry weight. G418 is most effective against dividing cells. By permitting cell growth prior to the addition of G418, one will maximize the selection characteristics of the antibiotic for both sensitive and resistant cells. Procedure: To generate a stable cell line expressing your protein of interest, you need to determine the minimum concentration of antibiotic required to kill your untransfected host cell line. Test a range of concentrations (at least 6) to ensure that you determine the minimum concentration necessary for your cell line. 1. Seed cells at approximately 20-25% confluency on the appropriate number of plates for each time plate and allow cells to adhere overnight. For cells that require higher densities for viability, increase the number of cells seeded. 2. The next day, substitute culture medium with medium containing varying concentrations of the antibiotic (i.e., test 0, 50, 100, 200, 400, 800, 1000 µg/mL). 3. Replenish the selective medium every 3-4 days. 4. Count the number of viable cells at regular intervals to determine that appropriate concentration of antibiotic that prevents the growth of untransfected cells. Select the concentration that kills the majority of the cells in the desired number of days, generally 7-10 day
  • QUESTION: Can you clarify G418 potency vs. units?
  • ANSWER: units/mg is exchangeable with µg/mg. Potency is always measured against a reference by the inhibition ring method. The inhibitory effect was measured on B. subtilis ATCC6633. Second, Potency is always measured against a reference by the inhibition ring method at beginning. The results come outin units/mg compared to the number of reference, so units/mg is considered as biological measurement. However since purity/content is proportional to potency in the most case, one wants to link potency to purity/assay measured by chemical methods such as HPLC because chemical measurement can give active and non-active parts quantitatively with much better reproducibility. One assumes the active part represents biological activity anis often called potency too. To link classic units/mg, potency is also expressed as µg/mg (be careful, units is not universal definition). For G418, units/mg is exchangeable with µg/mg since one did comparison already. Remember, it is a salt and there is no way to get 100% potency. Active part% is up to sulfate part change.
  • QUESTION: What is the working concentration for selection?
  • ANSWER: The general guideline is 400 µg / ml for selection and 200 µg / ml for maintenance are required for mammalian cells. Plant cells require 25-50 µg / ml for selection and 10 µg / ml for maintenance and bacteria 8-16 µg / ml for selection.
  • QUESTION: How do you prepare a stock solution?
  • ANSWER: The general guideline is 400 µg / ml for selection and 200 µg / ml for preparing stock solution Using an analytical balance weigh an appropriate amount of G418 powder (use formula below). Reconstitute aseptically by adding the powder to distilled water (pH 5.6 to 7.0) to the desired concentration and sterile filter. Required weight (mg) = Desired Volume (ml) x Desired Concentration (µg / ml) Lot Potency (µg / mg) NOTE: For G418, units/mg is exchangeable with µg/mg. Potency is always measured against a reference by the inhibition ring method. The inhibitory effect was measured on B. subtilis ATCC6633.
  • QUESTION: What is the working concentration for selection?
  • ANSWER: The working concentration for the purpose of selection varies with cell type, media, growth conditions and cell metabolic rate. Recommended concentration for the selection of resistant cells is 25-1000 µg/ml. Commonly used concentrations for selection are 200 µg/ml for mammalian cells, 20-200 µg/ml for plant cells & bacteria cells and 200-1000 µg/ml for fungi. Your optimum concentration should be tested experimentally .
  • QUESTION: How do I perform a Dose Response curve of G418 solution in Hepes buffer?
  • ANSWER: The outcome of this protocol is to produce a solution of G418 and hepes in the concentration of 500 mg of active G418 to 1 ml of hepes. 1. Find the active percentage of G418 as labeled on the vial by the producer (example 760 mg active per g or 76 percent active) 2. Mass the G418 in the vial and calculate the amount active G418 present (example mass of 1.178 mg with 76% active is 1.178*.76 = .89528 g active) 3. Determine the amount of hepes to be used by, dividing the amount of active G418 (in mg) by 500. This is the volume in ml of hepes that needs to be used to generate the desired concentration (example 895.28 mg G418 / 500 = 1.79 ml of hepes) 4. Prepare the solution in a 15 ml tube, filter and aliquot into microtubes in portions of no less the 500 microliters. 5. Store at 20ºC Thaw and add G418 at the appropriate concentration. The drug looses activity after one week, make new media with fresh drug as needed. Vials of G418 can be added to media to the following form to produce the given concentrations for finding the killing curve. for 50 mls of media 200 µg/ml need 20 µl 400 µg/ml need 40 µl 500 µg/ml need 50 µl 800/ml need 60 µl 800 µg/ml need 80 µl
  • QUESTION: Do you have any information on cell lines identifying concentrations for G-418 to create stable transfectants?
  • ANSWER: The values in brackets indicate the volume of antibiotic stock solution to add to 12 ml RPMI COMPLETE MEDIUM WHEN G418 stock is 10mg/ml. CELL LINE G-418 ALVA31 400 µg/ml (480 µl) PC3 100 µg/ml (120 µl) DU145 400 µg/ml (480 µl) JCA1 200 µg/ml(120 µl) LNCaP 200 µg/ml (240 µl)
  • QUESTION: How stable is material. Any concerns?
  • ANSWER: G418 solid is stable for up to 2 years, when kept closed and refrigerated at +4ºC. G418 Solutions should ideally be prepared fresh prior to use. Photo degradation over time is a possibility. Use opaque or amber bottle.
  • QUESTION: How there any hazard concerns?
  • ANSWER: Yes. It is considered hazardous material. Care should be taken to avoid contact with skin & eyes. Please consult & review Material safety data sheet (M.S.D.S.) prior to handling.
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  2. G-418 Sulfate Solution | CAS 108321-42-2 | Antibiotics | AG Scientific, Inc.
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