Hygromycin B / Paclobutrazol

Hygromycin B

CAS:31282-04-9

Molecular formula: c20h37n3o13

Molecular weight: 527.52

Purity: >90%

Physicochemical properties: white to almost white powder, easily soluble in water, methanol and ethanol.

Purpose: biochemical research

Mechanism of action: hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus metabolism, which kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis. Hygromycin B inhibits protein synthesis by interfering with 70s ribosomal translocation and inducing misreading of mRNA templates. At present, it is commonly used to screen and maintain prokaryotic or eukaryotic cells containing hygromycin resistance genes.

Resistance gene:

Resistance to hygromycin B is derived from the gene encoding hygromycin B phosphotransferase (HPH). So far, two sources have been found:

► HPH resistance gene produced by Streptomyces hygroscopicus;

► the HPH resistance gene carried by the endoplasmic granules of Escherichia coli, Klebsiella pneumoniae (commonly used);

Biological applications:

1) Screening and maintaining the culture of prokaryotic cells or eukaryotic cells (plant cells and mammalian cells) stably transfected with hph+ vector;

2) Due to the difference of action mode, it is often compared with G418 (geneticintm), zeocin ™  Combined with blasticidin s, cell lines stably transfected with two different vectors were screened;

3) Antiviral agent, hygromycin B selectively penetrates into cells with enhanced permeability due to virus infection, and has the effect of inhibiting translation;

4) Add animal feed as insect repellent;

Application concentration:

The working concentration of hygromycin B used to screen stable transgenic plants needs to change according to cell type, culture medium, growth conditions and cell metabolic rate. The recommended concentration is 50-1000 μ G/ml, the best concentration needs to be determined by the killing curve.

Mammalian cells · 200-500 μ g/mL； Bacteria / plant cells · 100-300 μ g/mL； Fungi · 300-1000 μ g/mL； 

Product use:

Use 1: kill curve to determine the best kill concentration (for reference only, which varies according to personal detection system)

(1) Untransfected cells with a cell density of about 50-200 cells / well were added to the 96 well plate of tissue culture level; After the cells adhere to the wall, add different concentrations (such as 50-1000) into each well μ G/ml, at least 5 concentrations) 200% of hygromycin B μ L fresh medium;

(2) Incubate cells in 5% CO2 incubator at 37 ℃ for 10-14 days;

(3) After 5-7 days of culture, replace with a new culture medium, which contains hygromycin B of corresponding concentration;

(4) After 10-14 days, cell proliferation methods such as MTT and CCK-8 were used to evaluate cell viability; Toxic effects can also be determined by detecting the number of cell clones or percentage confluence. Generally, the minimum concentration that can kill all cells in 10-14 days is the best screening concentration.

Use 2: screening stable transfected cells

(1) For adherent cells, directly suck the transfected cell culture medium in 60mm Petri dish, and then add 5-6ml of fresh culture medium containing hygromycin B; For suspended cells, the sterile condition was 250x g, centrifuged for 10min to remove the old transfected cell medium, and then suspended in about 5ml of fresh medium containing hygromycin B;

(2) After 5-7 days, replace the fresh medium containing hygromycin B as above;

(3) Incubate the cells for 5-7 days;

(4) After 10-14 days of incubation, the culture system only contains living cells that can express hygromycin B resistance phenotype. Therefore, after screening, as in step 1, replace the fresh medium without hygromycin B.