Puromycin

Description:

Protein synthesis inhibitors. Inhibit the growth of bacteria, algal protoorganisms and mammalian cells.

Puromycin is an aminoglycoside antibiotic produced by the fermentative metabolism of Streptomyces alboniger that kills Gram-positive bacteria, various animal and insect cells by inhibiting protein synthesis. Escherichia coli is effective in certain cases. The mechanism of action is that purinomycin is an analogue of the 6 'terminal of the aminoacyl-trNA molecule, which binds to the A site of the ribosome and is incorporated into the extended peptide chain. After binding to the A site, purinomycin does not participate in any subsequent reaction, resulting in early termination of protein synthesis and the release of immature peptides containing purinomycin at the C-terminal.

The pac gene found in the purinomycin producing bacterium Streptomyces alboniger encodes purinomycin n-acetyltransferase (PAC), which confers resistance to purinomycin. This feature is now commonly used to screen specific mammalian stably transfected cell lines carrying pac gene plasmids.

The widespread application of purinomycin in the screening of cell stable strains is related to the characteristics of lentiviral vectors, and most of the commercial lentiviral vectors carry pac gene. In some specific cases, purinomycin can also be used to screen for transformation of E. coli strains carrying pac gene plasmids.

Use and synthesis method:

Usage method

1. Recommended concentration

Mammalian cells: 1-10 μg/mL, the optimal concentration needs to be determined by killing curve;

Escherichia coli: LB AGAR medium was used to screen Escherichia coli with stable transformation of pac gene at a concentration of 125μg/mL. Note: Screening for stable E. coli strains using purinomycin requires precise pH regulation and is influenced by the host cell itself.

2. Dissolution method

Purinomycin was dissolved in distilled water to prepare 50 mg/ml mother liquor, which was filtered by 0.22 μm filter membrane and then subpackaged and frozen at -20℃. It can also be dissolved in methanol and formulated into 10 mg/ml storage solution.

3. Determination of the kill curve of purinomycin (using shRNA transfection or lentiviral transduction as examples)

The effective screening concentration of purinomycin is related to cell type, growth state, cell density, cell metabolism and cell cycle position. In order to screen for stably expressed shRNA cell lines, it is critical to determine the minimum concentration of purinomycin to kill untransfected/transduced cells. It is recommended that customers who do experiments for the first time must establish a kill curve suitable for their own experimental system.

1) Day 1:24 well plates were paved with a density of 5~8 x 104 cells/ holes, and enough holes were laid to carry out the subsequent gradient experiment. Cells were incubated overnight at 37℃.

2) Day 2: a) Prepare screening media: fresh media with different concentrations of purinomycin (e.g., 0-15μg/mL, at least 5 gradients); b) Replace the freshly prepared screening medium in the cells incubated overnight; Then the cells were incubated at 37℃.

3) Day 4: Replace the fresh screening medium and observe the cell survival rate.

4) According to the growth state of the cells, about 2-3 days to replace the fresh screening medium;

5) Cells are monitored daily to observe the survival rate to determine the minimum concentration of the drug to effectively kill non-transfected or all non-transduced cells within 4-6 days of antibiotic screening commencing.

4. Screening of stable transfected cell lines in mammals

After transfecting plasmids containing pac gene, cells were multiplied in a culture medium containing purinomycin to screen out stable transfectors.

1) 48h after transfection, the cells (as is or diluted) were cultured in a fresh medium containing appropriate concentrations of purinomycin.

Note: Antibiotics are most effective when cells are active in division. If the cells are too dense, the effectiveness of antibiotics will be significantly reduced. It is best to divide the cells so that their density does not exceed 25%.

2) Every 2-3 days, remove and replace the culture medium containing purinomycin.

3) After 7 days of screening, the cells formed lesions were evaluated. Lesions may require an additional week or more, depending on the host cell line and transfection screening efficiency. Note: Cell growth status should be observed daily. The screening of purinomycin takes at least 48 hours, and the screening cycle of effective concentration purinomycin is generally 3-10 days.

4) Transfer and place 5-10 resistant clones into a 35mm petri dish and continue to culture with selected medium for 7 days. This enrichment culture is to prepare for future cytotoxicity experiments.

Solubility:

Soluble in water, reference concentration 50mg/ml.

Note :

(1) For cell experiments, after dissolution, disposable needle filter must be used to remove bacteria.

(2) For your safety and health, please wear a lab coat and wear disposable gloves.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.

Remark：These protocols are for reference only. Solarbio does not independently validate these methods.

Note：

1. The products are all for scientific research use only. Do not use it for medical, clinical diagnosis or treatment, food and cosmetics, etc. Do not store them in ordinary residential areas.

2. For your safety and health, please wear laboratory clothes, disposable gloves and masks.

3. The experimental results may be affected by many factors, after-sale service is limited to the product itself and does not involve other compensation.