Puromycin

Manufactured by GoldBio

Sourced in United States

Puromycin is a laboratory reagent used as a selection marker in cell culture experiments. It functions by inhibiting protein synthesis, allowing for the identification and selection of cells that have successfully integrated a gene of interest.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Geneprint 10 system, by Promega (4 mentions) Polybrene, by Merck Group (4 mentions) Penicillin streptomycin, by Corning (3 mentions) Non essential amino acids (neaa), by Corning (3 mentions) L glutamine, by Corning (3 mentions) Pmd2 g envelope plasmid, by Addgene (3 mentions) Lenti x concentrator, by Takara Bio (3 mentions) Fugene 6, by Promega (3 mentions) Pspax2 packing plasmid, by Addgene (3 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Tr 1003 g, by Merck Group (2 mentions) Shnontarget, by Merck Group (2 mentions) Shprrx1 2, by Merck Group (2 mentions) Shprrx1, by GoldBio (2 mentions) Sodium pyruvate, by Corning (2 mentions) Ix70 microscope, by Olympus (2 mentions) Cycloheximide (chx), by Nacalai Tesque (1 mentions) Eq0001, by Kerafast (1 mentions)

Spelling variants of this product

Puromycin dihydrochloride (2 citations)

28 protocols using puromycin

Generating Lentiviral Knockdown of PRRX1 in PANC1 Cells

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Lentiviruses were produced in HEK293FT cells using the following shRNAs purchased from Sigma-Aldrich: shNonTarget
(SHC016), shPRRX1 #1 (TRCN0000020648) and shPRRX1 #2 (TRCN0000020646). Briefly, HEK293FT cells in a 10cm dish were transfected
using Lipofectamine 2000 with 10µg of shRNA plasmid and 5µg of the lentiviral packaging plasmids psPAX2 and pMD2.G.
Then, 48 hours following transfection the media containing lentiviruses was collected and filtered through a 0.45µm filter.
PANC1 cells were infected with the viral suspension containing 8μg/mL of polybrene (TR-1003-G, Millipore) for 1 hour at 5%
CO₂ and 37°C. Complete media containing 8μg/mL of polybrene was added and the cells were grown for 48
hours. Infected cells were selected (10 days) with 2μg/mL puromycin (Goldbio) for generation of stable PANC1-shCTL,
PANC1-shPRRX1 #1 and PANC1-shPRRX1 #2 populations.

Marchand B., Pitarresi J.R., Reichert M., Suzuki K., Laczkó D, & Rustgi A.K. (2019). PRRX1 isoforms cooperate with FOXM1 to regulate the DNA damage response in pancreatic cancer cells. Oncogene, 38(22), 4325-4339.

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Measuring Protein Synthesis with SUNsET

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To measure protein synthesis, we used the nonradioactive surface sensing of translation (SUNsET) technique. Briefly, 293A-EUA-EGFP cells were pre-treated with 10 μg/mL cycloheximide (Nacalai Tesque) or 10 μM IBT21 for 1–4 hr, followed by 2 μg/mL puromycin (GoldBio, MO, USA) treatment for 30 min. Total protein was extracted and immunoblot analysis was performed with an anti-puromycin antibody (EQ0001, Kerafast, MA, USA). Ponceau S (Sigma-Aldrich, MO, USA) was used as a loading control.

Kitakaze K., Taniuchi S., Kawano E., Hamada Y., Miyake M., Oyadomari M., Kojima H., Kosako H., Kuribara T., Yoshida S., Hosoya T, & Oyadomari S. (2019). Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity. eLife, 8, e43302.

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3D Culture of LOXL2-Expressing Breast Cancer Cells

Cited 3 times

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Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/ml D-glucose containing 10% fetal bovine serum and 1% penicillin and streptomycin, in a 5% CO2 incubator. MCF-7 cells were purchased from American Type Culture Collection (ATCC) and were stably transected with pCDNA3-LOXL2 [20 (link)] using ESCORT transfection reagent (Sigma–Aldrich Ltd.) and selected by puromycin (Gold Biotechnology). MCF-7-shLOXL2 cells were prepared as previously described [45 (link)]. D2.0R-LOXL2 cells were prepared by lentiviral infection of D2.0R cells using the NSPI-CMV-Myc lentiviral expression vector for human LOXL2 [46 (link)] and selection was performed by 2 μg/ml puromycin.
Three-dimensional cultures were carried out as previously described [13 (link), 25 ]. Briefly, eight-chamber glass slides (Lab -TEK^® II, Naperville, IL) were coated with 60 μl of growth factor reduced BME Cultrex^® (BME) (Trevigen Inc., Gaithersburg, MD). 5 × 10³ cells/well were resuspended in DMEM low glucose + 2% FBS supplemented with 2% BME.

Weidenfeld K., Schif-Zuck S., Abu-Tayeh H., Kang K., Kessler O., Weissmann M., Neufeld G, & Barkan D. (2016). Dormant tumor cells expressing LOXL2 acquire a stem-like phenotype mediating their transition to proliferative growth. Oncotarget, 7(44), 71362-71377.

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Stable Cdc42 FLARE cell lines

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Parental U2OS cells (HTB96; ATCC) and all derived clonal cell lines were cultured in Advanced DMEM supplemented with 10% FBS (Hyclone) and 2 mM GlutaMAX-I (GIBCO, Rockville, MD) at 37°C with 5% CO₂. Stable cell lines were generated using the PB transposon system (System Biosciences). In brief, U2OS cells were cotransfected with a PB-transposon construct (PB-mApple-p58, PB-GalT-mCherry, PB-ManII-mCherry, PB-mApple-Farnesyl, or PB-Cdc42-FLARE) and PB-transposase using X-tremeGENE 9 (Roche) as described by the manufacturer. Cells were selected for integration of the transposon using G418 at 500 µg/ml or puromycin at 2 µg/ml (Gold Biotechnology). Clonal cell lines were generated through serial dilution in 96-well plates.
Expression Cdc42-FLARE was accomplished by incubating the cells with 300 µg/ml water-soluble cumate (Systems Bio Sciences) for 48 h. For transient transfections, DNA constructs were introduced into cells using Lipofectamine 3000 (Life Technologies) or X-treme Gene 9 according to manufacturer’s protocol.

Herrington K.A., Trinh A.L., Dang C., O’Shaughnessy E., Hahn K.M., Gratton E., Digman M.A, & Sütterlin C. (2017). Spatial analysis of Cdc42 activity reveals a role for plasma membrane–associated Cdc42 in centrosome regulation. Molecular Biology of the Cell, 28(15), 2135-2145.

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Lentiviral Transduction and Puromycin Selection

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Cells were seeded at a density of 1 × 10⁵ cells per well in a 6-well tissue-culture treated plate and transduced overnight with lentiviral shRNAs Supplemented with polybrene (1 µg/mL; Santa Cruz, cat#sc-134220). Media was then removed and replaced with fresh media containing puromycin (2 µg/mL, Gold Bio, cat#P-600-1). After two days of selection, cells were counted and 1 × 10⁵ cells/well were plated in a 6-well plate, in triplicate. Three days later, cells were counted with trypan blue exclusion.

McKenzie L.D., LeClair J.W., Miller K.N., Strong A.D., Chan H.L., Oates E.L., Ligon K.L., Brennan C.W, & Chheda M.G. (2019). CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma. Scientific Reports, 9, 4444.

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Establishment and Characterization of Anaplastic Thyroid Cancer Cell Lines

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Anaplastic thyroid cancer cell lines (SW1736, 8505C, OCUT-2, and KTC-2) were cultured in RPMI 1640 growth media with L-glutamine (300 mg/L), sodium pyruvate and nonessential amino acids (1%) (Corning Inc, Corning, NY, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and penicillin-streptomycin (200 IU/L) (Corning) at 37°C, 5% CO₂, and 100% humidity. Lentivirally modified SW1736 cells were generated as recently described (11 ) with either an empty vector (SW-EV) or to overexpress TRβ (SW-TRβ). SW-EV and SW-TRβ were grown in the above conditions with the addition of 1 μg/ml puromycin (Gold Bio, St Louis, MO, USA). SW1736 and KTC-2 were authenticated by the Vermont Integrative Genomics Resource at the University of Vermont using short tandem repeat profiles and Promega GenePrint10 System (SW1736, May 2019; KTC-2, October 2019). 8505C and OCUT-2 were authenticated by University of Colorado by short tandem repeat profiles (8505C, June 2013; OCUT-2, June 2018). TheSW1736 and KTC-2 cells were tested for mycoplasma by PCR as described by Uphoff et al (SW1736, April 2019; KTC-2, February 2020; 85050C, February 2020; OCUt-2, January 2020) (13 (link)).

Bolf E.L., Gillis N.E., Davidson C.D., Rodriguez P.D., Cozzens L., Tomczak J.A., Frietze S, & Carr F.E. (2020). THYROID HORMONE RECEPTOR BETA INDUCES A TUMOR SUPPRESSIVE PROGRAM IN ANAPLASTIC THYROID CANCER.. Molecular cancer research : MCR, 18(10), 1443-1452.

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Yeast Growth Media and Selective Agents

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Yeast Extract Peptone Dextrose (YPD) rich media (2% glucose, 2% peptone, 1% yeast extract) and synthetic complete (SC) minimal media (2% glucose, 1× yeast nitrogen base; Research Products International, Mount Prospect, IL), with appropriate amino acid and base drop out compositions for selections were used (Formedium, Norfolk, UK). Rich media containing 1 mg/ml lead nitrate was prepared using a modified recipe (4% glucose, 0.3% peptone, 0.5% yeast extract, 0.02% ammonium sulfate). Geneticin (G418; Research products International, Mount Prospect, IL) was used at a concentration of 250 μg/ml in rich media. Puromycin (Gold Biotechnology, St. Louis, MO) was used at 4 mM for selection of strains carrying the pdr5Δ mutation and 20 mM for wild-type background strains. Methotrexate (Sigma-Aldrich, St. Louis, MO) was used at a final concentration of 25 nM in synthetic complete plates. To minimize the expense of Puromycin containing plates we routinely use 35 × 10 mm plates containing 2 mls of solidified agar media. Sulphanilamide (Fisher Scientific, Pittsburgh, PA) was added to Methotrexate containing plates at a final concentration of 5 mg/ml. 5-fluoroorotic acid (5-FOA; GoldBiotechnology, St. Louis, MO) was added to SC plates at a final concentration of 1 mg/ml. 5-fluoroanthranillic acid (5-FAA; Matrix Scientific, Columbia, SC) was used in SC media at a concentration of 0.5 mg/ml.

MacDonald C, & Piper R.C. (2015). Puromycin and Methotrexate Resistance Cassettes and Optimized cre-recombinase Expression Plasmids for use in Yeast. Yeast (Chichester, England), 32(5), 423-438.

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Lentiviral Transduction and Bioluminescent Tracking of Leukemia Cell Lines

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Both C1498 and FBL3 leukemia cell lines were transduced with lentivirus carrying the firefly luciferase (FLuc) transgene with puromycin resistance (Kerafast, FCT006; Boston, MA, USA). Forty-eight hours following transduction, cells were selected in RPMI media containing 5 µg/mL puromycin (ThermoFisher; Waltham, MA, USA) for 1–2 weeks. Bulk resistant cells were expanded in puromycin-containing RPMI and confirmed to be bioluminescent following addition of 200 µg/mL D-luciferin (GoldBio; St. Louis, MO, USA). Bioluminescence was detected using an iBright imaging system (Thermofisher; Waltham, MA, USA) or a Lago X live imager (Spectral Imaging; Tucson, AZ, USA).

Ebelt N.D, & Manuel E.R. (2022). 5-Azacytidine-Mediated Modulation of the Immune Microenvironment in Murine Acute Myeloid Leukemia. Cancers, 15(1), 118.

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Generation and Validation of GBM Cell Lines

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SJ-GBM2 cells (CVCL_M141) were a gift of the COG Repository at the Health Science Center, Texas Tech University (Lubbock, Texas, USA). SJ-GBM2 cells were grown in IMDM (Gibco, Thermo Fisher Scientific, catalog 1244005320) supplemented with 20% FBS at 37.0°C, 5% CO₂, 20% O₂ according to a previously published report (27 (link)) and were used in early passages and tested regularly for mycoplasma. SJ-GBM2 cells were transfected using jetPRIME (Polyplus, catalog 114-01) with the plasmids pLVX-BFP-H3.3WT and pLVX-BFP-H3.3G34R described above (Supplemental Figure 4). After transfection, cells were allowed to grow for 4 days, and selected with puromycin (10 μg/μL, Goldbio, catalog P-600-10). After amplification in the presence of selection, cells were subjected to FACS for isolation of BFP⁺ cells. Three independent polyclonal populations were obtained for each genotype (SJ-GBM-2-H3.3-G34R and SJ-GBM-2-H3.3-WT). These cells are referred to as H3.3-G34R and human pHGG cells.

Haase S., Banerjee K., Mujeeb A.A., Hartlage C.S., Núñez F.M., Núñez F.J., Alghamri M.S., Kadiyala P., Carney S., Barissi M.N., Taher A.W., Brumley E.K., Thompson S., Dreyer J.T., Alindogan C.T., Garcia-Fabiani M.B., Comba A., Venneti S., Ravikumar V., Koschmann C., Carcaboso Á.M., Vinci M., Rao A., Yu J.S., Lowenstein P.R, & Castro M.G. (2022). H3.3-G34 mutations impair DNA repair and promote cGAS/STING-mediated immune responses in pediatric high-grade glioma models. The Journal of Clinical Investigation, 132(22), e154229.

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Overexpression and Knockdown Experiments

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For overexpression experiments except in Figure 2G, cells were transfected using Lipofectamine2000 (Invitrogen; 11668019) according to the manufacturer’s instructions. For stable overexpression experiments in Figure 2G, cells were transduced with lentiviral particles expressing pLVX-EGFP or pLVX-EGFP-MISP. For KD experiments, cells were transduced with lentiviral particles expressing PLKO.1 scramble control and MISP-targeted shRNA plasmids (Sigma-Aldrich; TRCN0000422523, TRCN0000116527). For both stable overexpression and KD experiments, lentiviral particles were generated by transfecting HEK293T cells with 6 μg of the corresponding lentiviral expression vector alluded to above, 4 μg psPAX2 packing plasmid (Addgene, 12260), and 0.8 μg pMD2.G envelope plasmid (Addgene; #12259) using FuGENE 6 (Promega; E2691). Lentiviral particles were harvested and concentrated using a Lenti-X Concentrator (Clontech; 631231). Concentrated lentiviral particles were supplemented with polybrene (Sigma-Aldrich; H9268) and incubated with W4 cells at 80% confluency. After 24 h, the media was replaced with fresh media containing puromycin (Gold Biotechnology; P-600–100) for selection. Selection was applied for 14 days, replacing with fresh selection media every other day. For KD experiments, rescue assays were conducted using an EGFP-MISP construct designed to be refractory to shRNA KD.

Morales E.A., Arnaiz C., Krystofiak E.S., Zanic M, & Tyska M.J. (2022). Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli. Cell reports, 39(3), 110692.

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