Puromycin is an aminoglycoside antibiotic produced by fermentation and metabolism of Streptomyces alboniger, which kills Gram-positive bacteria, various animal and insect cells by inhibiting protein synthesis. Puromycin is effective against E. coli in some cases. The mechanism is that puromycin is an analogue of the 3´ terminal of aminoyl-TrNA molecules, which can bind to the A site of the ribosome and be incorporated into the extended peptide chain. After puromycin binds to the A site, it will not participate in any subsequent reactions, resulting in premature termination of protein synthesis and release of immature polypeptides containing puromycin at the C-terminus.

The pac gene found in the puromycin-producing strain Streptomyces alboniger encodes a puromycin N-acetyltransferase (PAC) that confers resistance to puromycin. This property is now commonly used to screen mammalian stably transfected cell lines carrying pac gene plasmids.

The general application of puromycin in the selection of stable cell transfections is related to the characteristics of lentiviral vectors. Most of the commercial lentiviral vectors now carry the pac gene. In some specific cases, puromycin can also be used to screen for the transformation of E. coli strains carrying the pac gene plasmid.

This product is suitable for cell culture, common working concentration is 1-10 µg/mL.

In addition, Yeasen company also provides puromycin (cat# 60209ES) in solution form, which is in a different state but has the same function.

Features

Purity≥98%

Applications

Suitable for cell culture

Screen specific mammalian stably transfected cell strains carrying plasmids carrying the pac gene

Screen the transformed E. coli strains carrying the pac gene plasmid

Specifications

Components

Shipping and Storage

The puromycin dihydrochloride products should be stored at -15℃ ~ -25℃ for 2 years.

Instructions

1. Suggested working concentration  

Mammalian cells: 1-10 μg/mL, optimum concentration needs to be determined by killing curve.  

Escherichia coli: LB agar medium was used to screen Escherichia coli stably transformed with pac gene at a concentration of 125 μg/mL.  

Note: Screening of stable E. coli strains using puromycin requires precise pH adjustment.

Recommended concentrations of puromycin hydrochloride

2. Dissolution method

Dissolve puromycin in distilled water to prepare a stock solution of 50 mg/mL. After sterilization by filtration through a 0.22 μm filter membrane, aliquot and store at -25 to -15℃. Alternatively, it can be dissolved in methanol to prepare a storage solution of 10 mg/mL.

3. Determination of puromycin killing curve (taking shRNA transfection or lentivirus transduction as an example)  

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

1) Day 1: The 24-well plate is plated at a density of 5-8×104 cells/well, and a sufficient number of wells are plated for subsequent gradient experiments. Cells were incubated overnight at 37℃.

2) Day 2: A) Prepare screening medium: fresh medium containing different concentrations of puromycin (such as 0-15 μg/mL, at least 5 gradients); B) Replace the freshly prepared screening medium in the cells after overnight incubation; Then the cells are incubated at 37℃.

3) Day 4: Replace with fresh selection medium and observe cell viability.

4) Depending on the growth state of the cells, change to fresh selection medium every 2-3 days.

5) Cells were monitored daily to observe the rate of viable cells to determine the lowest concentration of drug effective to kill non-transfected or all non-transduced cells within 4-6 days of the start of antibiotic screening.

4. Screening of Mammalian Stably Transfected Cell Lines

After transfection of the plasmid containing the pac gene, the cells were propagated in a medium containing puromycin to select stable transfectants.

1) 48 h after transfection, the cells (original or diluted) are cultured in fresh medium containing an appropriate concentration of puromycin.  

Note:Antibiotics are most effective when cells are actively dividing. If the cells are too dense, the effectiveness of antibiotics will be significantly reduced. It is best to plate cells to a density of no more than 25%.

2) Remove and replace the culture medium containing puromycin every 2-3 days.  

3) Cell-formed foci are assessed 7 days after screening. Lesions may require an additional week or more, depending on the host cell line and transfection screening efficiency.

Note:Observe the cell growth status every day. Screening of puromycin requires at least 48 h, and the screening period of effective concentration of puromycin is generally 3-10 days.

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

Documents:

Safety Data Sheet

60210_MSDS_HB220421_EN.pdf

Manuals

60210_Manual_HB250114_EN.pdf

Citations & References:

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