• Puromycin dihydrochloride packaged and labeled.

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SKU: P001

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Description

Puromycin Dihydrochloride (syn: Puromycin DiHCl) is the hydrochloride salt of Puromycin, an aminonucleoside antibiotic with anti-trypanosomal and antineoplastic properties.  Puromycin was isolated from Streptomyces alboniger in the 1950s.  Puromycin Dichloride is routinely used in cell culture as a selective agent in transfection and transformation protocols to select for cells that have been transformed with the pac gene and express puromycin-N-acetyl-transferase.  Puromycin DiHCl is soluble in water.

We also offer:

  • Puromycin (P097)
  • Puromycin Aminonucleoside (P041)
  • Puromycin DiHCl Solution (10 mg/ml in 20 mM HEPES)(P025-P026)

    CAS Number

    58-58-2

    Molecular Formula

    C22H29N7O5 · 2 HCl

    Molecular Weight

    544.43

    Mechanism of Action

    Puromycin Diihydrochloirde inhibits protein synthesis in two ways: 1) Puromycin associates with the donor substrate, peptidyl-tRNA, in the P site and functions as an acceptor substrate. 2) Purmycin DiHCl can compete with aminoacyl tRNA to bind with the A′ site within the peptidyl transferase center causing premature chain termination.

    Mechanism of resistance

    Resistance to Puromycin is conferred by the pac gene, a 60 nt fragment that encodes puromycin N-acetyltransferase. The enzyme inactivates Puromycin by acetylating the amino group in the tyrosinyl moiety. Acetylated Puromycin is biologically inactive and does not associate with prokaryotic or eukaryotic ribosomes.

    Storage Conditions

    2-8°C

    Tariff Code

    2941.90.1010

    Spectrum

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

Applications

    Cancer Applications

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

    Eukaryotic Cell Culture Applications

    Puromycin DiHCl has been used to screen cells 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. colii 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.

Specifications

    Form

    Powder

    Appearance

    White or off-white powder

    Water Content (Karl Fischer)

    ≤12.0%

    Melting Point

    168-178°C

References

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