Rapamycin

Manufactured by Research Products International

Rapamycin is a macrolide compound isolated from the bacterium Streptomyces hygroscopicus. It functions as an immunosuppressant and has applications in various fields of research, including cell biology, biochemistry, and pharmacology.

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Lab products found in correlation

Problock gold protease inhibitor cocktail, by GoldBio (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Avantor (2 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Cholera toxin, by List Biological Laboratories (1 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions) Puromycin, by Mirus Bio (1 mentions) Hygromycin b, by GoldBio (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Farnesyltransferase inhibitor, by Enzo Life Sciences (1 mentions) Insulin, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Dmem f12, by Corning (1 mentions) Rpmi 1640, by Corning (1 mentions) Mccoy s 5a, by Corning (1 mentions) Rpmi 1640 media, by Corning (1 mentions) Signalsilence control sirna unconjugated, by Cell Signaling Technology (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions) Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)

4 protocols using rapamycin

Quantifying Sch9 Phosphorylation in Yeast

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A plasmid expressing Sch9 under the ADH1 promoter (pJG1288) was made by amplifying Sch9-HIS using primers JG3330 and JG3331 and cloning into pJG1285. This Sch9 plasmid was transformed into yeast (WT [JGY1], reg1 [JGY95], snf1 [JGY91], reg1psk1psk2 [JGY283], reg1snf1psk1psk2 [JGY280]), grown in SD-Trp overnight, and then diluted 1:100 into YPAD and grown until OD₆₀₀ ∼1.0. For rapamycin controls, 100 nM rapamycin (Research Products International, Mount Prospect, IL) was added 1 h before harvesting cells. Preparation of Sch9 protein extract was performed as described by Miller-Fleming et al. (2014) (link). Samples were normalized and loaded on 8% SDS–PAGE, transferred to nitrocellulose membrane, and incubated overnight with anti–phospho-Thr737-Sch9 and anti–Thr737-Sch9 antibodies (Kingsbury et al., 2014 (link)). Intensity signals were quantified using ImageJ (National Institutes of Health, Bethesda, MD).

DeMille D., Badal B.D., Evans J.B., Mathis A.D., Anderson J.F, & Grose J.H. (2015). PAS kinase is activated by direct SNF1-dependent phosphorylation and mediates inhibition of TORC1 through the phosphorylation and activation of Pbp1. Molecular Biology of the Cell, 26(3), 569-582.

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CRISPR-based Point Mutation Introduction

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For editing of diverse genomic loci, HEK293T cells (lacking the single copy d2gfp) were used and maintained as above. The transfection protocol was performed as described above, with the exception that different sgRNAs were used for targeting of other loci. In each case, the sgRNAs expose a window where base editing can result in the introduction of point mutations in DNA modifying enzymes that lead to either missense or nonsense mutations. As with the d2GFP editing assay, 24 hours after transfection, rapamycin (Research Products International) was added to select wells at a final concentration of 200 nM. This concentration was continuously maintained until the end of the experiment. Transfected cells were harvested at day 3 after transfection, ensuring single-cell suspension. Genomic DNA was collected using the DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer’s instructions for sequencing analysis as described in the “DNA library preparation and sequencing” section below.

Berríos K.N., Evitt N.H., DeWeerd R.A., Ren D., Luo M., Barka A., Wang T., Bartman C.R., Lan Y., Green A.M., Shi J, & Kohli R.M. (2021). CONTROLLABLE GENOME EDITING WITH SPLIT-ENGINEERED BASE EDITORS. Nature chemical biology, 17(12), 1262-1270.

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Cellular Reagents and Inhibitors Database

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Farnesyltransferase inhibitor was obtained from Enzo Life Sciences (Farmingdale, NY) and geranylgeranyltransferase I inhibitor was from Tocris Biosciences (Bristol, UK). Horse serum and penicillin-streptomycin were from Life Technologies, Alexa Fluor 488 phalloidin and lipofectamine RNAiMAX transfection reagent were from Invitrogen, and EGF was from PeproTech, at Thermo Fisher Scientific (Waltham, MA). Hygromycin B and ProBlock gold protease inhibitor cocktail were obtained from Gold Biotechnology (St-Louis, MI). DMEM, DMEM/F12, RPMI-1640, and McCoy’s 5A media were from Corning (Corning, NY). Rapamycin was from Research Products International (Mount Prospect, IL); Torin2 was from ApexBio (Houston, TX); fetal bovine serum (FBS) was from Avantor (Radnor Township, PA); hydrocortisone was from United States Pharmacopeia (Rockville, MD); cholera toxin was from List Biological Laboratories (Campbell, CA); puromycin was from Mirus Bio LLC (Madison, WI); and insulin was from Sigma-Aldrich (St. Louis, MO).

Collins S.E., Wiegand M.E., Werner A.N., Brown I.N., Mundo M.I., Swango D.J., Mouneimne G, & Charest P.G. (2023). Ras-mediated activation of mTORC2 promotes breast epithelial cell migration and invasion. Molecular Biology of the Cell, 34(2), ar9.

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Examining Ras Signaling Pathways

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Life Technologies Penicillin-Streptomycin, Life Technologies OptiMEM I Reduced Serum Medium, Invitrogen Lipofectamine RNAiMAX Transfection reagent, PeproTech Animal-free Recombinant Human EGF, Invitrogen MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-Diphenyltetrosolium), and Corning RPMI 1640 Medium (Mod. w/o phenol red) 1X without glutamine, were purchased from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) and PP242 were obtained from Avantor (Radnor Township, PA); RPMI-1640 media was purchased from Corning (Corning, NY); Rapamycin was obtained from Research Products International (Mt. Prospect, IL); and ProBlock Gold Protease Inhibitor Cocktail was from Gold Biotechnology (St. Louis, MI). Dharmacon ON-TARGETplus Non-targeting siRNA Control Pool and SMARTPool of Human H-Ras (3265), Human N-Ras (4893), and Human K-Ras (3845) were obtained from Horizon Discovery (Waterbeach, UK), and SignalSilence Control siRNA (unconjugated) and SignalSilence Rictor siRNA were purchased from Cell Signaling Technology (Danvers, MA).

Werner A.N., Kumar A.I, & Charest P.G. (2023). CRISPR-mediated reversion of oncogenic KRAS mutation results in increased proliferation and reveals independent roles of Ras and mTORC2 in the migration of A549 lung cancer cells. Molecular Biology of the Cell, 34(13), ar128.

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