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Puromycin dihydrochloride (Synonyms: CL13900)

Catalog No.GC16384

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

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Puromycin dihydrochloride Chemical Structure

Cas No.: 58-58-2

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10mM (in 1mL DMSO)
$45.00
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20mg
$50.00
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50mg
$100.00
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100mg
$160.00
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500mg
$640.00
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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.

Product has been cited by 5 publications

Description of Puromycin dihydrochloride

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.

Protocol of Puromycin dihydrochloride

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 μg/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

0.5–6 μg/ml for 7 d

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.

Chemical Properties of Puromycin dihydrochloride

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 Water Storage Store at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table of Puromycin dihydrochloride

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1 mg 5 mg 10 mg
1 mM 1.8368 mL 9.1839 mL 18.3678 mL
5 mM 0.3674 mL 1.8368 mL 3.6736 mL
10 mM 0.1837 mL 0.9184 mL 1.8368 mL
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In vivo Formulation Calculator (Clear solution) of Puromycin dihydrochloride

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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

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    Puromycin dihydrochloride- GlpBio

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