Puromycin Dihydrochloride (DiHCl) solution is an aminonucleoside antibiotic solution derived from Streptomyces alboniger. Puromycin DiHCl is routinely used as a selective agent in transfection and transformation protocols.
Puromycin DiHCl solution is prepared at 10 mg/mL in 20 mM HEPES buffer.
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|Mechanism of Action||During translation, puromycin enters the ribosomal “A” site and disrupts peptide transfer. As a result, the ribosome stops and the peptide chain is terminated leading to a nonfunctional protein.|
|Spectrum||Puromycin dihydrochloride is active against both prokaryotic and eurkaryotic cells.|
|Eukaryotic Cell Culture Applications||Puromycin dihydrochloride is commonly used as a selective agent to isolate mammalian cells after transfection with the pac gene. The pac gene encodes puromycin N-acetyltransferase; a protein that inactivates puromycin by acetylation. |
For more information on relevant cell lines, media, and working concentrations, please visit the TOKU-E Cell Culture Database.
Azzam, M. E. "Mechanism of Puromycin Action: Fate of Ribosomes after Release of Nascent Protein Chains from Polysomes." PNAS 70.12 (1973): 3866-3869. www.ncbi.gov. Web. 4 Sept. 2012.
Vara, J. "Cloning and Expression of a Puromycin N-acetyl Transferase Gene from Streptomyces Alboniger in Streptomyces Lividans and Escherichia Coli." Gene 33.2 (1985): 195-206. Www.ncbi.gov. Web. 7 Sept. 2012.
Puromycin DiHCl Kill Curve Protocol
Puromycin DiHCl is an aminonucleoside antibiotic derived from Streptomyces alboniger and is routinely used as a selective agent for mammalian cells that have been transformed or transfected with plasmids containing the puromycin resistance gene, pac. Before stable transfected cell lines can be selected, the optimal puromycin DiHCl concentration needs to be determined by performing a kill curve titration. The optimal concentration of puromycin DiHCl suitable for selection of resistant mammalian clones depends on the cell lines, media, growth conditions, and the quality of puromycin DiHCl but typically lies between 1 µg/mL - 10 µg/mL. Because of these variables, it is necessary to perform a kill curve for every new cell type and new batch of puromycin DiHCl.
Preparation and storage of puromycin DiHCl solution:
Puromycin DiHCl is soluble in water at 50 mg/mL yielding a clear, colorless to faint yellow solution. The stock solution may be passed through a 0.22 μm filter and stored in aliquots at –20 °C.
Kill curve/puromycin DiHCl titration protocol:
Plasmid DNA Transfection Protocol
Once the appropriate antibiotic concentration to use for selection of the stable transfected cells has been determined by performing a kill curve, the next step is to generate a stable cell line by transfection of the parental cell line with a plasmid containing the gene of interest and an antibiotic resistance gene.
Plasmid DNA Transfection Protocol:
Seed 24-wells with insert and determine the transfection efficiency by immunostaining:
Selection of Stable Transfected Cell Lines Protocol
Once the cells have been successfully transfected, the next step is to seed and select the transfected cell line in a single 96-well plate to select pure colonies by limited dilution as outlined below:
Seed 24-wells with insert for an immunostaining to determine percentage of cells expressing the gene of interest to be able to identify a 100% pure clone. You can also use Western blotting, flow cytometry or another technique depending on the cell line used.
Seed 24-wells with insert and determine the expression level of the gene of interest by immunostaining:
|MIC||Streptococcus pneumonia (ATCC 6303 + quinolone-susceptible)| 1 － ?| 437| Streptococcus pneumonia (ATCC 7257 + quinolone-resistant)| 1 － ?| 437||