• Puromycin dihydrochloride packaged and labeled.

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Puromycin dihydrochloride (DiHCl) is an aminonucleoside antibiotic derived from Streptomyces alboniger. Puromycin DiHCl is routinely used in cell culture application as a selective agent in transfection and transformation protocols.

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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" 

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    C22H29N7O5 · 2 HCl

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    Mechanism of Action

    Puromycin Dihydrochloride inhibits protein synthesis in two ways. Puromycin associates with the donor substrate, peptidyl-tRNA, in the P site and functions as an acceptor substrate. Purmycin DiHCl can also compete with aminoacyl tRNA to bind with the A′ site within the peptidyl transferase center causing premature chain termination.

    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.

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    Puromycin dihydrochloride is active against both prokaryotic and eurkaryotic cells.  Puromycin DiHCl is active against gram-positive microorganisms, less active against acid-fast bacilli and gram-negative microorganisms. Puromycin can prevent growth of bacteria, algae, protozoa, and mammalian cells and acts quickly, killing 99% of cells within 2 days.


    Cancer Applications

    Puromycin DiHCl has shown anti-tumor activity when tested against numerous cell lines.

    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.
    Puromycin DiHCl has been used for the screening of successfully transfected cells with the CRISPR/Cas system, by co-transfecting a resistance plasmid, and screening with Puromycin as a selection marker.
    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 puromycin kill curve protocol, click here.

    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.





    White or off-white powder

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    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.

    Algire, Mikkel A. "New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species." Antimicrobial Agents and Chemotherapy 53.10 (2009): 4429-432. Ncbi.nlm.nih.gov. Web. 5 Nov. 2014.

    Lacalle R. et al., 1989. Molecular analysis of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger. Gene. 79:375-80.

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