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Puromycin dihydrochloride

Catalog No.GC16384

Puromycin dihydrochloride Chemical Structure

Puromycin dihydrochloride is produced by Streptomyces alboniger, a grampositive actinomycete, through a series of enzymatic reactions

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10mM (in 1mL DMSO)
$45.00
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20mg
$50.00
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50mg
$100.00
In stock
100mg
$160.00
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500mg
$640.00
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1g
$1,024.00
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Sample solution is provided at 25 µL, 10mM.

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Protocol

Cell experiment [1]:

Cell lines

Fetal porcine somatic cells

Preparation Method

Cells were seeded in 24-well plates at a density of 2.5 x 104 cells per well and cultured in medium containing 0.5–6 mg/ml puromycin dihydrochloride. Stock solution (10 mg/ml) of puromycin dihydrochloride was prepared by dissolving puromycin dihydrochloride in distilled water at the appropriate concentration. Media containing variable amounts of puromycin dihydrochloride were freshly prepared by adding the appropriate volume of puromycin dihydrochloride stock solution.

Reaction Conditions

Cells were incubated with puromycin for 7 days. Prepared Puromycin dihydrochloride was stored at 48 ℃. The concentration of puromycin dihydrochloride should be lower than 2 μg/ml.

Applications

Puromycin dihydrochloride is an antibiotic that inhibits growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses. To determine the optimal concentration of puromycin dihydrochloride for selecting EGFPac-transfected cells, a puromycin dihydrochloride resistance test was performed with fetal porcine somatic cells. The puromycin-resistant gene (termed pac) encoding puromycin N-acetyl transferase was isolated from Streptomyces aboniger. If pac is introduced and expressed in animal cells, the cells can survive in the presence of puromycin dihydrochloride.

Animal experiment [2]:

Animal models

Female FVB/N mice, 8–10 weeks old.

Preparation Method

Puromycin dihydrochloride was dissolved in 100 μl of PBS. Mice were housed under a 12-h light/dark cycle with ad libitum access to food and water unless otherwise stated. Mice were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) before all surgical procedures.

Dosage form

Puromycin dihydrochloride was intraperitoneal injected to mice with a concentration of 0.040 μmol/g.

Applications

The antibiotic puromycin dihydrochloride (a structural analog of tyrosyl-tRNA), and anti-puromycin antibodies could be used to detect the amount of puromycin incorporation into nascent peptide chains as well as to measure changes in protein synthesis in cell cultures.

References:

[1]. Watanabe S, Iwamoto M, et al. A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Biol Reprod. 2005 Feb;72(2):309-15.

[2]. Goodman CA, Mabrey DM, et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 2011 Mar;25(3):1028-39.

Background

Puromycin dihydrochloride is produced by Streptomyces alboniger, a grampositive actinomycete, through a series of enzymatic reactions.[1] Puromycin dihydrochloride included a nucleoside covalently bound to an amino acid, mimicking the 30 end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes.[2] It inhibits the growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses.[3]

In vitro study determined the optimal concentration of Puromycin dihydrochloride for selecting EGFPac-transfected cells by performing a Puromycin dihydrochloride resistance test. The puromycin-resistant gene (termed pac) encoding puromycin N-acetyl transferase was isolated from Streptomyces aboniger. If pac is introduced and expressed in animal cells, the cells can survive in the presence of Puromycin dihydrochloride. Results ahowed that it could successfully produce a somatically cloned transgenic piglet using recombinant cells obtained after gene transfer of a transgene (carrying both EGFP and pac expression units) and subsequent in vitro selection with a low concentration (2 mg/ml) of puromycin.[3]

In vivo study was conducted to determine the surface sensing of translation (SUnSET) technique could be used to measure the protein synthesis in whole tissues. Since there is currently an intense interest in identifying the molecular mechanisms that regulate skeletal muscle protein synthesis. It allows for the visualization and quantification of protein synthesis and eliminates the need for generating radioactive tissues/animals. This study also determined that the surface sensing of translation could detect relatively acute changes in protein synthesis in the absence of changes in rRNA as well as detect not only increases but also decreases in protein synthesis in vivo. [4]

References:
[1]. Tercero JA, Espinosa JC, Lacalle RA, Jiménez A. The biosynthetic pathway of the aminonucleoside antibiotic puromycin, as deduced from the molecular analysis of the pur cluster of Streptomyces alboniger. J Biol Chem 1996;271(3):1579–90.
[2]. Aviner R. et al. The science of puromycin: From studies of ribosome function to applications in biotechnology. Comput Struct Biotechnol J. 2020 Apr 24;18:1074-1083.
[3]. Watanabe S, Iwamoto M, et al. A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Biol Reprod. 2005 Feb;72(2):309-15.
[4]. Goodman CA, Mabrey DM, et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 2011 Mar;25(3):1028-39.

Chemical Properties

Cas No. 58-58-2 SDF
Synonyms CL13900
Chemical Name (S)-2-amino-N-((2S,3R,4S,5R)-5-(6-(dimethylamino)-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)-3-(4-methoxyphenyl)propanamide dihydrochloride
Canonical SMILES O[C@H]1[C@H]([C@@H](CO)O[C@H]1N2C3=NC=NC(N(C)C)=C3N=C2)NC([C@H](CC(C=C4)=CC=C4OC)N)=O.Cl.Cl
Formula C22H29N7O5.2HCl M.Wt 544.43
Solubility ≥ 27.2mg/mL in DMSO, ≥ 99.4mg/mL in H2O Storage Store at -20°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin

Proc Natl Acad Sci U S A2012 Jan 10;109(2):413-8.PMID: 22160674DOI: 10.1073/pnas.1111561108

Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

Active Ribosome Profiling with RiboLace

Cell Rep2018 Oct 23;25(4):1097-1108.e5.PMID: 30355487DOI: 10.1016/j.celrep.2018.09.084

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

Puromycin reactivity does not accurately localize translation at the subcellular level

Elife2020 Aug 26;9:e60303.PMID: 32844748DOI: 10.7554/eLife.60303

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast

BMC Microbiol2021 Apr 20;21(1):120.PMID: 33879049DOI: 10.1186/s12866-021-02185-3

Background: Translation is an important point of regulation in protein synthesis. However, there is a limited number of methods available to measure global translation activity in yeast. Recently, O-propargyl-puromycin (OPP) labelling has been established for mammalian cells, but unmodified yeasts are unsusceptible to puromycin.
Results: We could increase susceptibility by using a Komagataella phaffii strain with an impaired ergosterol pathway (erg6Δ), but translation measurements are restricted to this strain background, which displayed growth deficits. Using surfactants, specifically Imipramine, instead, proved to be more advantageous and circumvents previous restrictions. Imipramine-supplemented OPP-labelling with subsequent flow cytometry analysis, enabled us to distinguish actively translating cells from negative controls, and to clearly quantify differences in translation activities in different strains and growth conditions. Specifically, we investigated K. phaffii at different growth rates, verified that methanol feeding alters translation activity, and analysed global translation in strains with genetically modified stress response pathways.
Conclusions: We set up a simple protocol to measure global translation activity in yeast on a single cell basis. The use of surfactants poses a practical and non-invasive alternative to the commonly used ergosterol pathway impaired strains and thus impacts a wide range of applications where increased drug and dye uptake is needed.

Effects of tacrolimus on autophagy protein LC3 in puromycin-damaged mouse podocytes

J Int Med Res2020 Dec;48(12):300060520971422.PMID: 33322998DOI: 10.1177/0300060520971422

Objective: To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.
Methods: An in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha (LC3) expression were compared.
Results: Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group.
Conclusions: Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.

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