Hygromycin B

((2S,3'R,3aS,4S,4'S,5'R,6R,6'R,7S,7aS)-4-[(1R,2S,3R,5S,6R)-3-amino-2,6-dihydroxy-5-(methylamino)cyclohexoxy]-6'-[(1R)-1-amino-2-hydroxy-ethyl]-6-(hydroxymethyl)spiro[4,6,7,7a-tetrahydro-3aH-[1,3]dioxolo[4,5-c]pyran-2,2'-tetrahydropyran]-3',4',5',7-tetrol)

Purity ≥92.0% (solution and powder)

Molecular Biology Grade

Solution concentration is 50mg/ml in PBS Buffer.

Hygromycin B is an aminocyclitol with a wide spectrum of activity that kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis. It is produced by the bacterium Streptomyces hygroscopicus.  Hygromycin B strongly inhibits protein synthesis through a dual effect on mRNA translation. Like other aminoglycoside antibiotics, hygromycin B induces misreading of aminoacyl-tRNA by distorting the ribosomal A site (decoding center). Hygromycin B also affects the ribosomal translocation process.

Hygromycin solutions have been cell culture tested and are virtually free of all impurities.   This solution package is prepared as 50mg active Hygromycin B/ml solution in PBS buffer.

Store the powder desiccated at -20°C and solution between 2-8°C

Solutions are made by TOKU-E and powder by Goldbio Technology

Available in the Continental U.S. only.

Hygromycin B Protocols

Background
Hygromycin B is an aminoglycoside antibiotic which targets both prokaryotic and eukaryotic cells. Hygromycin B is freely soluble in water (50 mg/mL). Hygromycin B can be used to select for a resistance plasmid in numerous cell lines. Effective working concentrations range from 100 – 1000 µg/mL

Preparation and storage
Hygromycin B is soluble in water at >50 mg/mL. It is also soluble in methanol or ethanol. Solutions should be sterilized by filtration, not by autoclaving.

Hygromycin B solutions have been reported to lose activity on freezing. Since solutions are stable refrigerated, freezing should be avoided. Hygromycin B products should be stored as supplied at 2-8 °C. The dry solid is stable for at least five years if stored at 2-8 °C. Hygromcyin B solutions are stable as supplied for two years if stored at 2-8 °C.

Mammalian Cell Culture

Kill curve procedure
To express a recombinant protein of interest in mammalian cells, a mammalian expression vector containing the hyg or hph gene must be introduced into the mammalian host cell line. The hyg or hph marker gene allows for selection of stable transfectants using hygromycin B resistance.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 to ensure that you determine the minimum concentration necessary for your cell line.

1. Dilute cells in culture medium to 2.0 × 10^4 cells/ml. Add 4.0 ml of the cell dilution to each well of ten 6-well plates. For cells that require higher densities for viability, proportionally decrease the cell dilution. Allow cells to adhere overnight.

2. Prepare a 5.0 mg/ml solution of hygromycin B in distilled deionized water if the antibiotic is in powder form. If the antibiotic is in liquid form, make a 1:200 dilution in distilled deionized water. Make test dilutions of hygromycin B using the following dilution chart:

3. Substitute culture medium with medium containing the varying concentrations of hygromycin B.

4. Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth of untransfected cells. Select the concentration that kills the majority of the cells in the desired number of days, generally 7-10 days.

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