Puromycin Dihydrochloride

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Puromycin dihydrochloride is an aminonucleoside antibiotic derived from Streptomyces alboniger. Puromycin dihydrochloride is routinely used as a selective agent in transfection and transformation protocols.

Molecular Formula: C₂₂H₂₉N₇O₅ · 2 HCl

Molecular Weight: 544.43
    
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.
   
Mechanism of resistance
Puromycin resistance is conferred by the pac gene and is isolated from Streptomyces alboniger. The pac gene is a 600 nucleotide fragment of DNA and encodes puromycin N-acetyltransferase. Puromycin N-acetyltransferase inactivates puromycin by acetylating the amino group in the puromycin tyrosinyl moiety. Acetylated puromycin is biologically inactive and does not associate with prokaryotic or eurkaryotic ribosomes.

Spectrum
Puromycin dihydrochloride is active against both prokaryotic and eurkaryotic cells.

Application:

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.
 
Effective working concentrations for selection of puromycin resistant cells range from 0.5 – 10 µg/mL. The optimal working concentration of puromycin dihydrochloride for selection of resistant mammalian clones depends on the cell lines used, puromycin quality, media, growth conditions, cell density, cell metabolic rate, cell cycle phase, and the plasmid carrying the pac resistance gene. A kill curve should therefore be performed to determine the optimal working concentration for every experimental system and for every lot of puromycin dihydrochloride. Optimal selection concentrations of puromycin typically range from 0.5 µg/mL - 2 µg/mL for suspended cells and 2 µg/mL - 5 µg/mL for adherent cells.

For additional information regarding relevant cell lines, resistance plasmids, and culture media, please visit our cell culture database.

Microbiology Applications

Puromycin dihydrochloride can be used to select for puromycin resistant bacteria that have been transformed with the pac gene. Resistant E. coli transformants can be isolated on pH adjusted LB medium using a puromycin concentration of 100-125 µg/mL.

Puromycin dihydrochloride can also be used as a selectable marker in mollicute research and has been successfully used to select for various Mycoplasma species after transformation with the puromycin resistance gene (pac). Tetracycline is traditionally used as a selectable maker for Mycoplasma; however, puromycin does not have any clinical value, is a potent protein synthesis inhibitor, and can be used to screen for a wide range of puromycin resistant Mycoplasma spp. Because of its unique mechanism of action, there is a low possibility of spontaneous resistance to puromycin by a point mutation.

References

Conti et al. used puromycin DiHCl from TOKU-E to select for eGFP expressing A549 cells. "Polymeric Nanocarriers And Their Oral Inhalation Forumlations For The Regional Delivery of Nucleic Acids To The Lungs."

Sandoval-Jaime et al. used puromycin DiHCl from TOKU-E to select for stably transfected cells. "Recovery of murine norovirus and feline calicivirus from plasmids encoding EMCV IRES in stable cell lines expressing T7 polymerase."

Mutonga et al. used puromycin from TOKU-E to select for resistant cells transformed with a vector containing SUV39H2 (a histone methyltransferase) and a puromycin resistance gene. "Targeting Suppressor of Variegation 3-9 Homologue 2 (SUV39H2) in Acute Lymphoblastic Leukemia (ALL)."

Lu et al. used blasticidin S HCl and puromycin dihydrochloride from TOKU-E to select for transfected AS-B145 and BT-474 cells. "Ovatodiolide Inhibits Breast Cancer Stem/Progenitor Cells through SMURF2-Mediated Downregulation of Hsp27"