Hygromycin B Gold

Hygromycin B Gold: an ultrapure Hygromycin B, endotoxin tested.

Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. It kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis.

It has been reported to interfere with translocation ^[1] and to cause mistranslation at the 70S ribosome ^[2].

Resistance to Hygromycin B

Hygromycin B is used as a selective agent in molecular genetics experiments on a wide variety of eukaryotic and prokaryotic species ^[3-5].

The hph gene confers hygromycin-resistance to cells expressing it and many vectors carrying the hph gene are available within the scientific community.

Typically, mammalian cells are sensitive to Hygromycin B concentrations of 50-200 µg/ml, and bacteria to 50-100 µg/ml.

Protocol to facilitate the transition from using Hygromycin B to Hygromycin B Gold.

References:

1. Cabanas M. et al., 1978. Dual interference of Hygromycin B with ribosomal translocation and with Aminoacyl-tRNA recognition. Eur. J. Biochem. 87:21‑7.
2. Gonzales, A. et al., 1978. Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes. Biochem Biophys Acta 521:459‑69.
3. Gritz L. & Davies J., 1983. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179-88.
4. Cullen D. et al., 1987. Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph. Gene 57:21-6.
5. Santerre R. et al., 1984. Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells. Gene 30:147-56.

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Specifications

Product concentration: 100 mg/ml solution

CAS number: 31282-04-9

Quality Control: Each lot is thoroughly tested to ensure the absence of lot-to-lot variation.

Purity: ≥ 90% (HPLC)

Endotoxin level: < 0.5 EU/mg

Physicochemical characterization: pH, appearance

Cell culture tested: potency validated in hygromycin‑sensitive and hygromycin-resistant mammalian cell lines

Non‑cytotoxicity of trace contaminants: absence of long‑term effects confirmed in hygromycin‑resistant cells

Formula: C₂₀H₃₇N₃O₁₃, HCl

Molecular weight: 563.5

Structure:

Contents

Hygromycin B Gold (previously named HygroGold™) is supplied as a sterile filtered yellow solution at 100 mg/ml in HEPES buffer.

This product is available in three pack sizes:

• ant-hg-1: 10 x 1 ml (1 g)
• ant-hg-2: 20 x 1 ml (2 g) 
• ant-hg-5: 1 x 50 ml (5 g)

Hygromycin B Gold is shipped at room temperature.

Upon receipt, it should be stored at 4°C or -20°C.

SPECIAL HANDLING: Hygromycin B is a hazardous compound. Avoid contact with skin and eyes.

Details

WORKING CONCENTRATIONS

Hygromycin B Gold is normally used at a concentration of 200 μg/ml, a 500-fold dilution from the stock solution. However, the optimal concentration needs to be determined for your cells.
Suggested concentrations of Hygromycin B Gold for selection in some examples of mammalian cells are listed below:

References:

1. Neerincx A. et al., 2012. NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner. J Immunol. 188(10):4940-50.
2. Saccheri F. et al., 2010. Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity. Sci Transl Med. 2(44):44ra57.
3. Hidmark A. et al., 2012.  Cutting edge: TLR13 is a receptor for bacterial RNA. J Immunol. 189(6):2717-21.
4. Hennen S. et al.,  2013. Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist. Sci Signal. 6(298):ra93.
5. Raschi E. et al., 2014. β-2-glycoprotein I, lipopolysaccharide and endothelial TLR4: Three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun. S0896-8411(14)00065-1.
6. Ravid D. et al., 2010. A role for caveolin 1 in assembly and budding of the paramyxovirus parainfluenza virus 5. J Virol. 4(19):9749-59.
7. Hayashi Y. et al., 2014. Mechanism by which a LINE protein recognizes its 3’ tail RNA. Nucleic Acids Res. 42(16):10605-17.
8. Bowen WS. et al., 2012. Selective TRIF-dependent signaling by a synthetic Toll-like receptor 4 agonist. Sci Signal. 5(211):ra13.
9. Leskela TT. et al., 2012. Cys-27 variant of human {delta}-opioidreceptor modulates maturation and cell surface delivery of Phe-27 variant via heteromerization. J. Biol. Chem., 287: 5008-20.
10. van den Bosch MW. et al., 2014. LPS induces the degradation of programmed cell death protein 4 (PDCD4) to release Twist2, activating c-Maf transcription to promote interleukin-10 production. J Biol Chem. 289(33):22980-90.
11. Benjamin LR. et  al.,  2008.  Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein. PNAS. 105(47):18296-301.
12. Bouwman LI. et al., 2014. Inflammasome Activation by Campylobacter jejuni. J Immunol. 193(9):4548-57.
13. Bryan NB. et al., 2009. Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J Immunol. 182(5):3173-82.

FAQs

In the TDS it is stated that less Hygromycin B Gold (50-100 µg/ml) can be used in low salt LB, however we only have premixed high salt LB in our lab, therefore what concentration of Hygromycin B Gold should be used for selection?
We recommend to use Hygromycin B Gold at a concentration between 100 – 150 µg/ml for high salt LB conditions.

What is the preferred storage method for Hygromycin B Gold?
Hygromycin B Gold is shipped at room temperature. Upon receipt, it can be stored at 4°C for short term storage or -20°C for long term storage.
Avoid repeated freeze-thaw cycles. The expiry date is specified on the product label.
Once opened it remains stable for 1 year at -20°C providing you avoid repeated freeze-thaw cycles, and it can be kept at 4°C for 3 months provided it is kept in sterile conditions.

What is the selection concentration of Hygromycin B Gold in mammalian cells?
The working concentration of Hygromycin B Gold in mammalian cell lines varies between 50 -200 μg/ml, with a few reported cases of up to 500 μg/ml. In an initial experiment we recommend to determine the optimal concentration of Hygromycin B Gold required to kill your host cell line. Generally, Hygromycin B Gold is used at 200 μg/ml, a 500-fold dilution of the stock solution.
For more detailed information, please refer to the technical data sheet for this product.

What is the selection concentration of Hygromycin B Gold in E. coli?
Hygromycin-resistant transformants are selected in low salt LB agar medium supplemented with 50 to 100 μg/ml Hygromycin B Gold. Plates containing Hygromycin B Gold are stable for 1 month when stored at 4 °C.

Any questions about our Selection antibiotics ? Visit our frequently asked questions page