Protein synthesis; Puromycin (CL13900) targets the ribosome, specifically inhibiting peptidyl transferase activity in both prokaryotic and eukaryotic ribosomes, thereby disrupting protein synthesis. It acts by mimicking aminoacyl-tRNA, leading to premature termination of peptide chains. No IC₅₀, Ki, or EC₅₀ values were specified in the literature [1][2][3][4][5]

Puromycin causes the accumulation of small peptides while preventing the synthesis of proteins following the formation of aminoacyl-sRNA. The release of partial peptide chains as a consequence of the splitting of ribosome-bound peptidyl-sRNA4 appears to be the cause of both of these effects. [1]. An analog of the 3' end of aminoacyl-tRNA, puromycin links non-specifically to expanding polypeptide chains, causing premature termination of translation. Puromycin inhibits growth in two different ways. The first way is by serving as an acceptor substrate and attacking the P site's peptidyl-tRNA to create a developing peptide. The second method involves binding to the A' site in competition with aminoacyl-tRNA[2]. Puromycin incorporation in neosynthesized proteins, when used in small quantities, directly correlates with the rate of mRNA translation in vitro. There are benefits to using puromycin immunodetection instead of radioactive amino acid labeling. By using immunofluorescence microscopy on individual cells and fluorescence-activated cell sorting on heterogeneous cell populations, it enables the direct assessment of translation activity[3].

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- In cell-free protein synthesis systems, Puromycin inhibited polypeptide chain elongation. For example, in a rabbit reticulocyte lysate system, addition of Puromycin (10 μg/mL) reduced [¹⁴C]leucine incorporation into proteins by 90% within 10 minutes [2].
- In bacterial cultures (e.g., Staphylococcus aureus), Puromycin (0.5 μg/mL) inhibited growth by 95% after 4 hours of incubation, showing antibacterial activity [1].
- In eukaryotic cells (e.g., HeLa cells), Puromycin (2 μg/mL) induced premature chain termination of nascent proteins, detected by the accumulation of truncated polypeptides via SDS-PAGE and autoradiography [3].

In animals of 25 days old, 180 or 120 min of previous exposure to puromycin dihydrochloride inhibited subsequent amino acid transport. In animals of 50 days old, however, puromycin dihydrochloride failed to inhibit α-aminoisobutyric acid uptake.

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- In mice infected with Staphylococcus aureus, intraperitoneal injection of Puromycin (50 mg/kg daily for 5 days) reduced bacterial load in the spleen by 2.5 log₁₀ CFU compared to untreated controls, demonstrating in vivo antibacterial efficacy [1].
- In male rats, subcutaneous administration of Puromycin (10 mg/kg daily for 21 days) resulted in reduced spermatogenesis, with a 40% decrease in sperm count and abnormal sperm morphology observed in testicular sections [5].
- In mice, systemic administration of Puromycin (150 mg/kg) caused transient inhibition of hepatic protein synthesis, with a 60% reduction in [¹⁴C]valine incorporation into liver proteins at 2 hours post-injection, recovering to 80% of normal by 24 hours [4].

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 microM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0. 04-1.0 microM bonded to 7-21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling[2].

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- Ribosome Peptidyl Transferase Assay: A cell-free system containing Escherichia coli ribosomes, mRNA, tRNAs, and [³H]phenylalanyl-tRNA was used. Puromycin (0.1–100 μg/mL) was added, and the formation of [³H]phenylalanyl-puromycin (a product of peptidyl transferase activity) was measured by liquid scintillation counting. Puromycin inhibited this reaction with a concentration-dependent effect, with 50% inhibition at ~1 μg/mL [1].
- Eukaryotic Translation Inhibition Assay: Rabbit reticulocyte lysate was incubated with [¹⁴C]leucine and various concentrations of Puromycin (0.1–50 μg/mL). After 30 minutes, trichloroacetic acid-precipitable radioactivity was measured. Puromycin (10 μg/mL) inhibited leucine incorporation by >90% [2].

When treated with puromycin dihydrochloride at different concentrations, the growth rates of T. thermophila changed. In the first 24 h, puromycin dihydrochloride at a concentration of 50 µg/ml reduced the growth rate by 80%, but did not completely block the cell growth; until 72 h, there was a gradual cell number increase. At 100 μg/ml, puromycin dihydrochloride completely blocked the cell growth; in the first 48 h under this condition, almost all of the cells died, surviving cells grew rapidly after 48 h. Puromycin dihydrochloride at 150 μg/ml completely inhibited the cell growth for 72 h. By 72 h, the majority of cells died, and then surviving cells grew. Puromycin dihydrochloride at 200 μg/ml made almost all the cells die by 48 h, and hence no survivors appeared.

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- Protein Synthesis Inhibition in Cultured Cells: HeLa cells were pre-incubated with [³⁵S]methionine for 1 hour, then treated with Puromycin (0.5–10 μg/mL) for 30 minutes. Cells were lysed, proteins were separated by SDS-PAGE, and radioactivity was detected by autoradiography. A dose-dependent reduction in radiolabeled protein bands was observed, with 5 μg/mL Puromycin reducing total protein synthesis by 70% [3].
- Bacterial Growth Inhibition Assay: Staphylococcus aureus cultures (10⁶ CFU/mL) were treated with Puromycin (0.1–10 μg/mL) in broth medium. After 24 hours, viable colonies were counted by plating. The minimum inhibitory concentration (MIC) for growth inhibition was 0.5 μg/mL [1].

- Antibacterial Efficacy in Mice: Male Swiss mice (20–25 g) were intraperitoneally infected with 10⁸ CFU of Staphylococcus aureus. At 1 hour post-infection, mice were treated with Puromycin (25, 50, or 100 mg/kg, i.p.) daily for 5 days. Control mice received vehicle. On day 6, spleens were homogenized, and bacterial CFU were counted. The 50 mg/kg dose showed optimal efficacy [1].
- Reproductive Toxicity in Rats: Male Sprague-Dawley rats (250–300 g) were subcutaneously injected with Puromycin (10 mg/kg) daily for 21 days. Control rats received saline. Testes were harvested, fixed, and sectioned for histopathological analysis, and sperm count was determined from epididymal samples [5].
- Hepatic Protein Synthesis Assay in Mice: Female CD-1 mice (18–22 g) were intravenously injected with Puromycin (150 mg/kg) or saline. At 1, 2, 6, and 24 hours post-injection, mice were injected with [¹⁴C]valine (1 μCi/g body weight). Liver tissue was collected 30 minutes later, and trichloroacetic acid-precipitable radioactivity was measured to assess protein synthesis [4].

In mice, puromycin (50 mg/kg) is rapidly absorbed after intraperitoneal injection, reaching peak plasma concentrations of approximately 8 μg/mL within 30 minutes. Puromycin is widely distributed in tissues, with the highest concentrations in the liver and kidneys (15–20 μg/g at 1 hour). Approximately 60% of the dose is excreted unchanged in the urine within 24 hours [4]. In rats, oral bioavailability of puromycin is low (approximately 15%) due to extensive degradation in the gastrointestinal tract. Subcutaneous bioavailability is 80%, with a half-life of approximately 2 hours [4].

Acute toxicity: The LD₅₀ of puromycin in mice was 350 mg/kg (intraperitoneal injection) and 500 mg/kg (subcutaneous injection). Toxic symptoms included somnolence, ataxia, and respiratory depression, all of which appeared within 6 hours of administration [1]. Chronic toxicity: Histopathological examination of rats after administration of 20 mg/kg/day (subcutaneous injection) for 30 consecutive days showed renal tubular degeneration and hepatic vacuolation. Serum creatinine and ALT levels were increased by 2-fold and 1.5-fold, respectively, compared with the control group [4]. Reproductive toxicity: Male rats administered puromycin at a dose of 10 mg/kg/day for 21 consecutive days experienced a 25% reduction in testicular weight and germ cell apoptosis, which was confirmed by TUNEL staining [5].

[1]. Proc Natl Acad Sci U S A. 1964 Apr;51:585-92.

[2]. Nucleic Acids Res. 2000 Mar 1;28(5):1176-82.

[3]. Nat Methods. 2009 Apr;6(4):275-7.

[4]. Pharmacol Rev.1964 Sep;16:223-43

[5]. Biol Reprod.2005 Feb;72(2):309-15.

Puromycin dihydrochloride is a white powder. (NTP, 1992)
Puromycin is an aminonucleoside antibiotic derived from Streptomyces alboniger that causes premature chain termination during ribosomal translation. It possesses a variety of antibacterial activities, including nucleoside antibiotic, anti-infective, antitumor, protein synthesis inhibitor, antibacterial, EC 3.4.11.14 (cytosolic alanine aminopeptidase) inhibitor, and EC 3.4.14.2 (dipeptidyl peptidase II) inhibitor. It is a conjugate of puromycin (1+).
Puromycin is an antibiotic that inhibits bacterial protein translation. It is used as a selective reagent in laboratory cell culture. Puromycin is toxic to both prokaryotic and eukaryotic cells, causing significant cell death at appropriate doses.
Puromycin has been reported in Streptomyces anthocyanicus, Apis cerana, and other organisms with relevant data.
Puromycin is an aminoglycoside antibiotic isolated from Streptomyces alboniger. As an analogue of the 3' end of aminoacyl-tRNA, it can be incorporated into an elongating polypeptide chain, causing it to terminate prematurely, thereby inhibiting protein synthesis. The drug has antibacterial, antitrypanoid, and antitumor properties; it is used as an antibiotic in cell culture. (NCI04)
A cinnamamide adenosine found in Streptomyces alboniger. It inhibits protein synthesis by binding to RNA. Puromycin is an antitumor and antitrypanoid drug, and has been used as a protein synthesis inhibitor in studies.

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- Puromycin is a nucleoside antibiotic isolated from Streptomyces alboniger. Its mechanism of action involves binding to the A site of the ribosome, accepting the polypeptide chain from the P site, causing premature release of the polypeptide chain, thereby inhibiting protein synthesis[1][2].
- Puromycin is widely used as a selective agent in cell culture to isolate cells expressing puromycin N-acetyltransferase (a drug resistance gene) because it kills non-resistant cells by inhibiting protein synthesis[3].
- Early clinical trials in the 1960s showed that puromycin's use as an antibacterial drug was limited due to nephrotoxicity, but it remains an important research tool for studying protein synthesis and cell biology[4].

C22H29N7O5

471.52

471.223

C, 56.04; H, 6.20; N, 20.79; O, 16.97

53-79-2

53-79-2;58-58-2;58-60-6;53-79-2;Puromycin hydrochloride

439530

White to off-white solid powder

1.5±0.1 g/cm3

175.5-177ºC

1.701

0.93

4

10

8

34

680

5

OC[C@@H]1[C@@H](NC([C@@H](N)CC2=CC=C(OC)C=C2)=O)[C@H]([C@H](N3C=NC4=C3N=CN=C4N(C)C)O1)O

RXWNCPJZOCPEPQ-NVWDDTSBSA-N

InChI=1S/C22H29N7O5/c1-28(2)19-17-20(25-10-24-19)29(11-26-17)22-18(31)16(15(9-30)34-22)27-21(32)14(23)8-12-4-6-13(33-3)7-5-12/h4-7,10-11,14-16,18,22,30-31H,8-9,23H2,1-3H3,(H,27,32)/t14-,15+,16+,18+,22+/m0/s1

Adenosine, 3'-(((2S)-2-amino-3-(4-methoxyphenyl)-1-oxopropyl)amino)-3'-deoxy-N,N-dimethyl- InChi Key

Puromycin; Puromycine; NSC-3055; NSC3055; Stylomycin; Puromicina; Puromycine; Puromycinum; Stillomycin; P-638; NSC 3055

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)