Puromycin

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Puromycin is a laboratory product manufactured by Merck Group. It functions as an antibiotic that inhibits protein synthesis in eukaryotic cells.

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Lab products found in correlation

5 590 protocols using puromycin

Lentiviral Transduction of Keratinocytes

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Cultured keratinocytes were infected as previously described (Beronja et al., 2010 (link)). Briefly, keratinocytes were plated at 10⁵ cells/well in six-well plates and infected with ∼10⁷ colony-forming units of the appropriate lentiviruses encoding control (shScr;puromycin or shScr;H2B-GFP) or gene-specific shRNA in the presence of 100 µg/ml Polybrene (Sigma-Aldrich).
For Western blot and cell proliferation analyses, cultured keratinocytes were infected with shScr;puromycin (control) or gene-specific shRNA;puromycin. 48 h after infection, cells were selected with 2 µg/ml puromycin (Sigma-Aldrich) for an additional 72 h and then processed (see below). Keratinocytes were cultured in 50 µM Ca²⁺; 24 h before harvesting, Ca²⁺ levels were increased to 300 µM. For differentiation assays, cultured keratinocytes were switched into 1.5 mM Ca²⁺ for 96 h and then analyzed.

Cohen J., Raviv S., Adir O., Padmanabhan K., Soffer A, & Luxenburg C. (2019). The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt–Sox9 signaling. The Journal of Cell Biology, 218(4), 1390-1406.

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Puromycin Detection in Oxidatively Stressed Trophozoites

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Trophozoites (2 × 10⁶/ml) were grown with and without queuine (0.1 μM for 3 days at 37°C) and were exposed or not to 2.5 mM H₂O₂ for 20 min at 37°C. After treatment with H₂O₂, the trophozoites were incubated with 10 μg/ml puromycin (Sigma) for 20 min. For pretreatment of the trophozoites with cycloheximide (Sigma), the trophozoites were incubated with 100 μg/ml cycloheximide for 5 min before the addition of puromycin. The trophozoites were lysed with 1% IGEPAL (Sigma) in phosphate-buffered saline (PBS) with protease inhibitors. puromycin was detected by immunoblotting as described above with a 12D10 clone monoclonal puromycin antibody (Millipore). The amount of total protein in each lane was determined by using the No-Stain protein labeling reagent (Thermo Fisher Scientific). Imaging for puromycin and total protein signals was performed on a FUSION FX7 EDGE imaging system (Witec AG). Quantification of signal density was performed using ImageJ.

Nagaraja S., Cai M.W., Sun J., Varet H., Sarid L., Trebicz-Geffen M., Shaulov Y., Mazumdar M., Legendre R., Coppée J.Y., Begley T.J., Dedon P.C., Gourinath S., Guillen N., Saito-Nakano Y., Shimokawa C., Hisaeda H, & Ankri S. (2021). Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator. mBio, 12(2), e03549-20.

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SUnSET Assay for Protein Synthesis Measurement

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SUnSET was performed using a previously described protocol (Hertz et al., 2014b (link); Shahi et al., 2016a (link)). Briefly, trophozoites (2 × 10⁶/ml) were incubated with 10 μg/ml puromycin (Sigma-Aldrich, St. louis, Mo, USA), a structural analog of tyrosyltRNA, for 20 min at 37°C. The trophozoites were lysed using 1% Igepal (Sigma) in PBS. Whole proteins were resolved on a 10% SDS-PAGE in SDS-PAGE running buffer. Proteins were electrotransferred in protein transfer buffer to nitrocellulose membranes. Loading equivalency was determined by immunoblotting using a 1:10,000 monoclonal α-tubulin antibody (DM1A clone, Sigma-Aldrich, St. louis, Mo, USA). puromycin was detected by immunoblotting using a 1:5,000 monoclonal puromycin antibody (12D10 clone, Millipore). After incubation with the primary antibody, the blots were incubated with 1:5,000 secondary antibody for 1 h at RT (Jackson ImmunoResearch, Enco Diagnostics, Israel), and then developed using enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, ThermoFisher Scientific, USA). Protein quantification/synthesis was estimated from the intensity of the immunoreactive blots (densitometry) using Fiji software (Schindelin et al., 2012 (link)).

Trebicz-Geffen M., Shahi P., Nagaraja S., Vanunu S., Manor S., Avrahami A, & Ankri S. (2017). Identification of S-Nitrosylated (SNO) Proteins in Entamoeba histolytica Adapted to Nitrosative Stress: Insights into the Role of SNO Actin and In vitro Virulence. Frontiers in Cellular and Infection Microbiology, 7, 192.

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Measuring Protein Synthesis in Sciatic Nerves

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Distal segments (1 cm long) of sciatic nerves from wild-type mice at 4 months, or control and RaptorKO mice 2 months after tamoxifen administration were collected, transferred to DMEM-Glutamax (Invitrogen) supplemented with 5% FCS (Invitrogen), and incubated at 37°C 5% CO₂ until ready for analysis. For Western blot analysis, the epineurium was removed immediately after dissection. To evaluate the rate of protein synthesis, a modified version of a puromycin-based assay was used. In this assay, puromycin is added cotranslationally to nascent polypeptides allowing to monitor the overall rate of protein synthesis (Schmidt et al., 2009 (link)). Briefly, nerve explants were incubated with puromycin (Sigma-Aldrich, 10 μg/ml) for 30 min immediately after dissection and removal of the epineurium (control), or after a 24 h treatment with DMSO or Torin 1 (250 nm). After incubation with puromycin, nerves were snap frozen, lysed, and subjected to Western blot analysis using an anti-puromycin antibody (Millipore, catalog #MABE343, RRID:AB_2566826, 1:1000) to detect newly synthesized proteins. Ponceaus S staining was used as loading control.

Norrmén C., Figlia G., Pfistner P., Pereira J.A., Bachofner S, & Suter U. (2018). mTORC1 Is Transiently Reactivated in Injured Nerves to Promote c-Jun Elevation and Schwann Cell Dedifferentiation. The Journal of Neuroscience, 38(20), 4811-4828.

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Axon Protein Synthesis Inhibition Assay

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After 48 hours, axons were severed from their cell bodies and subsequently treated with 2 μM puromycin (Sigma-Aldrich) for 10 minutes. After treatment, the cultures were fixed in 2% formaldehyde/7.5% sucrose in PBS for 20 minutes at RT, washed 3 times in PBS + 0.001% Triton-X-100, permeabilized for 3-5 minutes with 0.1% Triton-X-100 and blocked in 5% heat-inactivated goat serum in PBS for 30 minutes at RT and subsequently labeled with Alexa Fluor 647-conjugated mouse anti-puromycin antibody (1:250, MABE343-AF647, Millipore) overnight. For experiments involving pharmacological treatments, axons were pre-treated with 50μM CHX (Sigma-Aldrich) for 20 minutes, 50μM dynasore (Sigma-Aldrich) for 20 minutes, 50μM Chloroquine (Sigma-Aldrich) or 2.5μM PP242 for 10 minutes before puromycin administration.

Cioni J.M., Lin J.Q., Holtermann A.V., Koppers M., Jakobs M.A., Azizi A., Turner-Bridger B., Shigeoka T., Franze K., Harris W.A, & Holt C.E. (2019). Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons. Cell, 176(1-2), 56-72.e15.

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Puromycin Incorporation Assay in Giardia

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SUnSET was performed using a previously described protocol [23 (link)]. Briefly, trophozoites (2x10⁶/ml) that were treated with 2.5 mM H₂O₂ for 15 minutes at 37°C and untreated control trophozoites were incubated with 10 μg/ml puromycin (Sigma), a structural analog of tyrosyltRNA, for 20 minutes. For pretreatment of the trophozoites with cycloheximide (Sigma), the trophozoites were incubated with 100 μg/ml cycloheximide for five minutes before adding puromycin. The trophozoites were lysed using 1% Igepal (Sigma) in PBS. puromycin was detected by immunoblotting using a monoclonal puromycin antibody (12D10 clone, Millipore). Protein quantification was measured by band intensity (densitometry) using ImageJ software [17 (link)].

Shahi P., Trebicz-Geffen M., Nagaraja S., Alterzon-Baumel S., Hertz R., Methling K., Lalk M, & Ankri S. (2016). Proteomic Identification of Oxidized Proteins in Entamoeba histolytica by Resin-Assisted Capture: Insights into the Role of Arginase in Resistance to Oxidative Stress. PLoS Neglected Tropical Diseases, 10(1), e0004340.

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Measuring Zebrafish Muscle Protein Synthesis

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The protein synthesis rate in zebrafish skeletal muscle was measured according to the principles of the surface sensing of translation method as described previously (19) . Briefly, after the last meal, three fish from each tank were randomly selected and immediately exposed to 0•04 mmol/l puromycin (Sigma-Aldrich) for 2 h. Then, the dorsal muscle was sampled to quantify the puromycin-labelled peptides, which can be measured by Western blotting assay using a specific primary antibody against puromycin (clone 12D10, Cat. no. MABE343-AF647, Millipore).

Wang L., Yin J., Liao C., Cheng R., Chen F., Yu H, & Zhang X. (2023). Selenium deficiency-induced high concentration of reactive oxygen species restricts hypertrophic growth of skeletal muscle in juvenile zebrafish by suppressing TORC1-mediated protein synthesis. The British journal of nutrition, 130(11).

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Stable CD147 Knockdown in Synovioblasts

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The Trans-Lentiviral pLKO System was used to construct the stable CD147 knockdown synovioblasts RASF A6, and RASF NC was RASF transfected with lentivirus without the CD147 short hairpin RNA (shRNA) sequence. The construction of the lentivirus carrying CD147 shRNA was described previously [27 (link)]. Namely, the plasmids of Trans-Lentiviral pLKO System (psPAX2 1.125 μg/mL, pMD2G 0.125 μg/mL, pLKO A6/NC 1.25 μg/mL) were transfected into HEK 293T cells, and the medium (containing lentivirus) of HEK 293T cells was collected and then cultured with RASFs. Puromycin at 4 μg/mL (Sigma, Buchs, Switzerland) was added to the medium for further selection (10 days), and the successfully transfected RASF A6 and RASF NC cells were resistant to Puromycin, while the blank control group RASF CTL cells were killed by Puromycin at this concentration. To obtain stable cell clones, RASF A6 and RASF NC cells were maintained in medium containing 1 μg/mL Puromycin for at least 2 weeks.

Zhai Y., Wu B., Li J., Yao X.Y., Zhu P, & Chen Z.N. (2015). CD147 promotes IKK/IκB/NF-κB pathway to resist TNF-induced apoptosis in rheumatoid arthritis synovial fibroblasts. Journal of Molecular Medicine (Berlin, Germany), 94(1), 71-82.

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Jasplakinolide Treatments for Cytoskeleton Dynamics

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For in vivo jasplakinolide treatments, wild-type embryos were collected on E15.5 and incubated with 3μM jasplakinolide (Sigma-Aldrich), 30 nM Calyculin A (Cell Signaling Technology), or DMSO in serum-free DMEM (Biological Industries) at 37°C for 2 h before embedding in OCT or processed for whole-mount preparation and immunofluorescence microscopy as described above. For in vitro treatments, keratinocytes were infected with shScr;puromycin or shAnln;puromycin, selected with 3 μg/ml puromycin (Sigma) and plated on fibronectin-coated coverslips (40,000 cells in a single well of a 24-well plate). Twenty-four hours later, the medium was switched to high calcium (1.5mM Ca²⁺) and cells were treated with 100nM jasplakinolide (Sigma-Aldrich) or 2nM Calyculin A for 5 min, and then with 8μM nocodazole for 6 h. Cells were then fixed and labeled with Phalloidin-iFluor 647 (Abcam, ab176759) and pERM (Cell Signaling Technology, 1:200) overnight at 4°C. After washing, sections were incubated with a secondary antibody (1: 500 dilution) at room temperature for 1 h. Data was collected using a Nikon C2+ 60×/1.4 objective that generates optical sections of 0.49 μm at the middle of the cell.

Mahly A., Padmanabhan K., Soffer A., Cohen J., Omar J., Sagi-Eisenberg R, & Luxenburg C. (2022). Anillin governs mitotic rounding during early epidermal development. BMC Biology, 20, 145.

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In Situ Detection of Protein Synthesis

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Puromicyn is a tRNA analog, which is incorporated into the nascent polypeptide chain in a ribosome-catalyzed reaction. This technique allows the in situ detection of protein synthesis with an anti-puromicyn antibody. At 10 DIV, DMSO- and Aβ-treated neurons were exposed to 2 μM puromycin (Sigma Aldrich) for 5–30 min as indicated. Control conditions with no puromicyn received only fresh growth medium (vehicle). To verify that puromycin labels newly synthesized proteins, 40 μM of the translation inhibitor anisomycin (Sigma Aldrich) was co-incubated with puromycin. Cells were washed with cold PBS with 3 μg.ml^–1 digitonin (Sigma Aldrich) and fixed in 4% paraformaldehyde (PFA), 4% sucrose in PBS.

Gamarra M., Blanco-Urrejola M., Batista A.F., Imaz J, & Baleriola J. (2020). Object-Based Analyses in FIJI/ImageJ to Measure Local RNA Translation Sites in Neurites in Response to Aβ1-42 Oligomers. Frontiers in Neuroscience, 14, 547.

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