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Selection Antibiotics
Gibco high-quality selection agents offer unique solutions for your research needs, such as dual selection and rapid stable cell line establishment.
View our full list of antibiotics and antimycotics. In addition, explore the use of antibiotics in cell culture.
On this page:
- Eukaryotic and bacterial selection antibiotics
- G418, Geneticin
- Zeocin
- Puromycin
- Blasticidin
- Hygromycin B
Eukaryotic selection antibiotics
| Selection antibiotic | Most common selection usage | Common working concentration | Available powder sizes | Available liquid sizes |
|---|---|---|---|---|
| Blasticidin | Eukaryotic and bacteria | 1–20 µg/mL | 50 mg | 10 x 1 mL, 20 mL |
| Geneticin (G-418) | Eukaryotic | 100–200 µg/mL bacteria 200–500 µg/mL mammalian cells | 1 g, 5 g, 10 g, 25 g | 20 mL, 100 mL |
| Hygromycin B | Dual-selection experiments and eukaryotic | 200–500 µg/mL | — | 20 mL |
| Mycophenolic acid | Mammalian and bacteria | 25 µg/mL | 500 mg | — |
| Puromycin | Eukaryotic and bacteria | 0.2–5 µg/mL | — | 10 x 1 mL, 20 mL |
| Zeocin | Mammalian, insect, yeast, bacteria, and plants | 50–400 µg/mL | — | 8 x 1.25 mL, 50 mL |
Bacterial selection antibiotics
| Selection antibiotic | Most common selection usage | Common working concentration | Available powder sizes | Available liquid sizes |
|---|---|---|---|---|
| Actinomycin D | Bacteria | 1 µg/mL | 5 mg, 10 mg | — |
| Ampicillin sodium salt | Bacteria | 10–25 µg/mL | 200 mg | — |
| Blasticidin | Eukaryotic and bacteria | 50–100 µg/mL | 50 mg | 10 x 1 mL, 20 mL |
| Carbenicillin, disodium salt | Agrobacterium and E. coli | 100–500 µg/mL | 5 g | — |
| Kanamycin sulfate | Bacteria | 100 µg/mL | 5 g, 25 g | 100 mL |
| Mycophenolic acid | Mammalian and Bacteria | 25 µg/mL | 500 mg | — |
| Neomycin sulfate | Bacteria | 50 µg/mL | 100 g | — |
| Polymyxin B sulfate | Bacteria | 100 units/mL | 25 MU | — |
| Puromycin | Eukaryotic and bacteria | 0.2–5 µg/mL | — | 10 x 1 mL, 20 mL |
| Streptomycin sulfate | Bacteria | 50–100 µg/mL | — | 100 g |
| Zeocin | Mammalian, insect, yeast, bacteria, and plants | 75–400 µg/mL | — | 8 x 1.25 mL, 50 mL |
Eukaryotic selection antibiotics
| Selection antibiotic | Most common selection usage | Common working concentration | Available powder sizes | Available liquid sizes |
|---|---|---|---|---|
| Blasticidin | Eukaryotic and bacteria | 1–20 µg/mL | 50 mg | 10 x 1 mL, 20 mL |
| Geneticin (G-418) | Eukaryotic | 100–200 µg/mL bacteria 200–500 µg/mL mammalian cells | 1 g, 5 g, 10 g, 25 g | 20 mL, 100 mL |
| Hygromycin B | Dual-selection experiments and eukaryotic | 200–500 µg/mL | — | 20 mL |
| Mycophenolic acid | Mammalian and bacteria | 25 µg/mL | 500 mg | — |
| Puromycin | Eukaryotic and bacteria | 0.2–5 µg/mL | — | 10 x 1 mL, 20 mL |
| Zeocin | Mammalian, insect, yeast, bacteria, and plants | 50–400 µg/mL | — | 8 x 1.25 mL, 50 mL |
Bacterial selection antibiotics
| Selection antibiotic | Most common selection usage | Common working concentration | Available powder sizes | Available liquid sizes |
|---|---|---|---|---|
| Actinomycin D | Bacteria | 1 µg/mL | 5 mg, 10 mg | — |
| Ampicillin sodium salt | Bacteria | 10–25 µg/mL | 200 mg | — |
| Blasticidin | Eukaryotic and bacteria | 50–100 µg/mL | 50 mg | 10 x 1 mL, 20 mL |
| Carbenicillin, disodium salt | Agrobacterium and E. coli | 100–500 µg/mL | 5 g | — |
| Kanamycin sulfate | Bacteria | 100 µg/mL | 5 g, 25 g | 100 mL |
| Mycophenolic acid | Mammalian and Bacteria | 25 µg/mL | 500 mg | — |
| Neomycin sulfate | Bacteria | 50 µg/mL | 100 g | — |
| Polymyxin B sulfate | Bacteria | 100 units/mL | 25 MU | — |
| Puromycin | Eukaryotic and bacteria | 0.2–5 µg/mL | — | 10 x 1 mL, 20 mL |
| Streptomycin sulfate | Bacteria | 50–100 µg/mL | — | 100 g |
| Zeocin | Mammalian, insect, yeast, bacteria, and plants | 75–400 µg/mL | — | 8 x 1.25 mL, 50 mL |
G418, Geneticin
Commonly known as G418 or G-418, Gibco 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.
Ordering information for G418, Geneticin
What's in your G418?
To be effective, selection agents need to be reliable, highly pure, and consistent in nature. Geneticin selective antibiotic, considered a reagent of choice 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 not related to purity. The potency value is a measure of bacterial growth inhibition.
- 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 ED50 value.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
ED50 assay 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 narrower 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 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 not related to purity. The potency value is a measure of bacterial growth inhibition.
- 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 ED50 value.
Therefore, comparing potency values between different suppliers is not a relevant specification for mammalian cell selection.
ED50 assay 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 narrower 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 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.
| 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) | 2,450–2,700 | 1,350–3,100 | 600–2,350 | 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. 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.
Zeocin
Gibco zeocin is highly effective in a variety of organisms, including mammalian and insect cell lines, as well as in yeast, bacteria, and plants. As a member of the bleomycin family, zeocin causes cell death by intercalating into and cleaving DNA.
Resistance to zeocin is conferred by the Sh ble gene product, which binds the antibiotic and prevents it from binding DNA. This selection agent is effective in multiple cell types, so eukaryotic expression vectors only need to carry one drug selection marker. This reduces the overall size of the vector and makes subcloning and transfection easier and more efficient.
Formula: C55H83N19O21S2Cu
Ordering information for zeocin
| Cell types | Conc. (μg/mL)* | Invitrogen vectors carrying resistance marker |
|---|---|---|
| 293 HEK | 200–400 | InsectSelect System, pIZ/V5-His vectors (stable expression in insect cells) |
| CHO | ~250 | pSecTag2, pSecTag2/Hygro (secreted mammalian expression) |
| COS-1 | ~400 | ZeoCassette vectors (constructing Zeocin-resistant vectors) |
| HeLa | <~150 | pcDNA4 vectors (constitutive mammalian expression) |
| Jurkat T cell | ~200 | pPICZ and pPICZ alpha vectors (inducible expression in Pichia pastoris) |
| NIH3T3 | ~400 | T-REx System, pcDNA4/TO vectors (inducible mammalian expression) |
| Pichia pastoris | ~100 | |
| S2 Drosophila | ~75 | |
| Sf9 insect | ~250 | pGAPZ and pGAPZ alpha vectors (inducible expression in Pichia pastoris) |
* The optimal concentration for selection of your cell line should be determined using kill curves.
Puromycin
Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. During puromycin selection, this antibiotic inhibits cellular protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the puromycin N-acetyltransferase gene (pac) from Streptomyces.
Puromycin selection at low concentrations
Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations. Adherent mammalian cells are sensitive to concentrations of 2 to 5 µg/mL, while cells in suspension are sensitive to concentrations as low as 0.5 to 2 µg/mL. Puromycin-resistant stable mammalian cell lines can be generated in less than one week.
Ordering information for puromycin
| Cell line | Concentration [µg/mL] | Reference |
|---|---|---|
| A549 (lung cancer) | 1.5 | Mol Cell Biol 27:324–339 (2007) |
| 1.5 | J Biol Chem 283:33394–33405 (2008) | |
| H1299 (non-small cell lung carcinoma) | 1 | Nucleic Acids Res 35:e17 (2007) |
| 1.5 | J Biol Chem 283:33394–33405 (2008) | |
| 2.5 | PNAS 105:1937–1942 (2008) | |
| HEK293 (human embryonic kidney) | 0.25 | Nucleic Acids Res 36:e83 (2008) |
| 2 | Mol Cell Biol 28:4104–4115 (2008) | |
| 3 | Mol Cell Biol 27:4708–4719 (2007) | |
| HeLa (human cervical cancer) | 1–2 | Genes Cells 12:397–406 (2007) |
| 2 | Mol Cell Biol 28:4104–4115 (2008) | |
| 2 | PNAS 105:16490–16495 (2008) | |
| Hep G2 (human hepatocellular carcinoma) | 0.3 | J Biol Chem 283:21462–21468 (2008) |
| 2 | Mol Cell Biol 28:4104–4115 (2008) | |
| 2 | J Biol Chem 283:708–715 (2008) | |
| HT1080 (human fibrosarcoma) | 0.2–0.4 | J Biol Chem 282:29314–29322 (2007) |
| 1 | J Virol 82:3320–3328 (2008) | |
| 2 | Cancer Res 68: 1417–1426 (2008) | |
| Human embryonic stem (ES) cells | 0.5 | Nucleic Acids Res 36:e148 (2008) |
| 1 | Stem Cells 26:850–863 (2008) | |
| 5 | Stem Cells 26:2245–2256 (2008) | |
MCF-7 (human breast cancer) | 0.45 | RNA 13:1375-1383 (2007) |
| 1 | Cancer Res 68:1319–1328 (2008) | |
| 2 | Mol Cell Biol 28:4104–4115 (2008) | |
| MDA-MB-231 (human breast cancer) | 0.5 | Mol Cell Biol 28:997–1006 (2008) |
| 1.5 | Mol Cell Biol 27:324 – 339 (2007) | |
| 5 | Mol Cell Biol 28:687–704 (2008) |
Blasticidin
Blasticidin is a nucleoside antibiotic produced by the bacterium Streptomyces griseochromogenes. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells. Resistance to blasticidin is conferred by the product of the bsd gene from Aspergillus terreus.
Blasticidin selection at low concentrations
Blasticidin has a fast, potent mode of action, causing rapid cell death at low antibiotic concentrations. E. coli strains are generally sensitive to concentrations of 50 µg/mL, while mammalian cells are sensitive to concentrations as low as 2 to 10 µg/mL. Cell death occurs rapidly, and blasticidin-resistant stable mammalian cell lines can be generated in less than one week.
Ordering information for blasticidin
| Cell Line | Concentration [ug/mL] | Reference |
|---|---|---|
| HT1080 (human fibrosarcoma) | 1–5 10 20 | J Biol Chem 283:11556–11564 (2008) J Virol 82:7325–7335 (2008) Mol Biol Cell 19:8–16 (2008) |
| HeLa (human cervical cancer) | 10 10 20 | J Gen Virol 89:2611–2621 (2008) Mol Biol Cell 19:8–16 (2008). J Immunol 181:22–26(2008) |
| HEK293 (human embryonic kidney) | 5 10 | J Biol Chem 283:27534–27546 (2008) J Virol 82:1665–1678 (2008) |
| Hep G2 (human hepatocellular carcinoma) | 4 5 | J Biol Chem 283:16320–16331 (2008) Blood 113: 1786–1793 (2009) |
| HT1080 (human fibrosarcoma) | 1–5 10 20 | J Biol Chem 283:11556–11564 (2008) J Virol 82:7325–7335 (2008) Mol Biol Cell 19:8–16 (2008) |
| MCF-7 (human breast cancer) | 2 5 | Mol Cancer Res 6:555–567 (2008) Mol Endocrinol 22: 361–379 (2008) |
| A549 (lung cancer) | 10 | Cancer Res 68:5040–5048 (2008) |
Hygromycin B
Hygromycin B is an aminoglycosidic antibiotic that inhibits protein synthesis by disrupting translocation and promoting mistranslation at the 80S ribosome. Because it uses a different mode of action than geneticin, blasticidin S, or zeocin, it’s useful for dual-selection experiments when used in conjunction with another selection agent.
Hygromycin selection genes and concentrations
Resistance to hygromycin B is conferred by the E. coli hygromycin resistance gene (hyg or hph). The concentration for selection ranges from 100–1,000 µg/mL (typically 200 µg/mL) and should be optimized for each cell line.
Formula: C20H37N3O13
FW: 527.5 g/mole
Ordering information for hygromycin
| Cell types | Conc. (μg/mL)* | Invitrogen vectors carrying resistance marker |
|---|---|---|
| CHO | ~250 | pIND/Hygro vector (Ecdysone-inducible mammalian expression) |
| HeLa | ~550 | pcDNA5 vectors (constitutive mammalian expression) |
| Jurkat T cell | ~1,000 | pSecTag2/Hygro vector (secreted mammalian expression) |
| S2 Drosophila | 200–300 | pREP vectors (episomal mammalian expression) |
| pCoHygro (selection vector for DES) |
* The optimal concentration for selection of your cell line should be determined using kill curves.
For Research Use Only. Not for use in diagnostic procedures.
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