Trametinib

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Trametinib is a small molecule inhibitor of mitogen-activated protein kinase kinase (MEK) 1 and MEK2. It is used in laboratory research to study the role of the MEK/ERK signaling pathway in various cellular processes.

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Lab products found in correlation

Paclitaxel, by Fujifilm (2 mentions) Mk 2206, by Santa Cruz Biotechnology (2 mentions) Gefitinib, by Santa Cruz Biotechnology (2 mentions) Pazopanib, by Selleck Chemicals (1 mentions) Pcr single locus technology, by Eurofins (1 mentions) Mycoalert plus, by Lonza (1 mentions) Cell lysis reagent, by Merck Group (1 mentions) Normal rabbit igg, by Cell Signaling Technology (1 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Anti ddk magnetic beads, by OriGene (1 mentions) Myelin basic protein, by Merck Group (1 mentions) Flag peptide, by Merck Group (1 mentions) Dynabeads protein a, by Thermo Fisher Scientific (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Doxorubicin, by Abcam (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Nutlin 3, by Selleck Chemicals (1 mentions) Rpmi medium, by Thermo Fisher Scientific (1 mentions) Ku 55933, by Santa Cruz Biotechnology (1 mentions) Tae684, by Santa Cruz Biotechnology (1 mentions)

8 protocols using trametinib

Small Molecule Drug Preparation

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Pazopanib was purchased from SelleckChem (Houston, TX, USA), BMS754807 from Active Biochemicals (Wanchai, Hong Kong, China), and trametinib from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For in vitro studies, drugs were dissolved in DMSO and further diluted in cell culture medium (0.25–0.5% DMSO final concentration).

Lanzi C., Dal Bo L., Favini E., Tortoreto M., Beretta G.L., Arrighetti N., Zaffaroni N, & Cassinelli G. (2019). Overactive IGF1/Insulin Receptors and NRASQ61R Mutation Drive Mechanisms of Resistance to Pazopanib and Define Rational Combination Strategies to Treat Synovial Sarcoma. Cancers, 11(3), 408.

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Investigating Cell Line Responses to MEK Inhibitor and Iron

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RKO, SW480, HCT116 and CACO‐2 cells (ACTT) were grown in DMEM with 10% (v/v) FCS, 50 U/mL penicillin and 50 μg/mL streptomycin. HT29 cells were cultured in McCoy's 5A medium. Cell authentication was performed in October 2016 by PCR‐single‐locus‐technology (eurofins, Germany) and cells were routinely tested for mycoplasma infection (MycoAlert PLUS, Lonza, Rockland, ME, USA). Prior to experimentation, cells were seeded into 6‐well plates at a concentration of 1 × 10⁵ cells/mL and incubated for 24 h. The MEK inhibitor, trametinib (Santa‐Cruz Biotechnology, Dallas, TX, USA), was supplemented into media at concentrations of 10 nM and 1 μM. Iron (FeSO₄∙7H₂O) (Sigma Aldrich, Dorset, England) was supplemented at a concentration of 100 μM with sodium ascorbate (500 μM). Epidermal growth factor (EGF) was utilized at 25 ng/mL.

Horniblow R.D., Bedford M., Hollingworth R., Evans S., Sutton E., Lal N., Beggs A., Iqbal T.H, & Tselepis C. (2017). BRAF mutations are associated with increased iron regulatory protein‐2 expression in colorectal tumorigenesis. Cancer Science, 108(6), 1135-1143.

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Investigating Protein Interactions and Signaling

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Antibodies against MEK2 (catalog no. sc-524) and EF1α (catalog no. 28578) were purchased from Santa Cruz Biotechnology, and normal rabbit IgG (catalog no. 2729) was from Cell Signaling Technology. Anti-FLAG M2 affinity gel (catalog no. A2220) was purchased from Sigma. Anti-DDK magnetic beads (catalog no. TA150042) were purchased from OriGene Technologies. FLAG peptide (catalog no. F3290) was purchased from Sigma. Lipofectamine 3000 transfection reagent (catalog no. L3000-001) was acquired from Life Technologies. MEK1/2 inhibitor U0126 (catalog no. 9903) was purchased from Cell Signaling Technology, while the MEK1/2 inhibitor trametinib (catalog no. sc-364639) was obtained from Santa Cruz Biotechnology. Dynabeads protein A (catalog no. 10002D) were purchased from Life Technologies. Cell lysis reagent (catalog no. C2978) for mammalian cells was from Sigma. Human total tRNA (catalog no. BH401) was purchased from Bio S&T Inc, or gel purified from HEK293T cells. Human total RNA was isolated from HEK293T cells. Inactive human ERK2 (catalog no. 14-536) was obtained from EMD Millipore. Myelin basic protein (catalog no. 13-104) was purchased from Millipore and Myelin basic protein derived peptide (catalog no. sc-3011) was from Santa Cruz Biotechnology. P81 phosphocellulose squares (catalog no. 20-134) were obtained from Whatman.

Wang X., Chow C.R., Ebine K., Lee J., Rosner M.R., Pan T, & Munshi H.G. (2016). Interaction of tRNA with MEK2 in pancreatic cancer cells. Scientific Reports, 6, 28260.

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Agar Preparation for Cell Cultures

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Low molecular weight (Ultra-agar Ina, average MW 43,000) and standard (S-6, average MW 290,000) agar was purchased from Ina Food Industry Co., Ltd. (Nagano, Japan). To prepare agar stock solution for cell cultures, agar was suspended in pure water to 1.0% (w/v) and dissolved by stirring at 90 °C. The aqueous solution was sterilized at 121 °C for 20 minutes by autoclave. The solution was then added to cell culture media at given concentrations with stirring at room temperature.
Trametinib and MK-2206 were obtained from Santa Cruz (Texas, USA). Paclitaxel was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Abe-Fukasawa N., Otsuka K., Aihara A., Itasaki N, & Nishino T. (2018). Novel 3D Liquid Cell Culture Method for Anchorage-independent Cell Growth, Cell Imaging and Automated Drug Screening. Scientific Reports, 8, 3627.

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Cell Line Characterization and Transfection

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293T (human embryonic kidney), DLD-1, HCT-116 (colorectal carcinoma), MCF7, and BT474 (breast cancer) cells were obtained from ATCC; PC9 cells (NSCLC) were obtained from ECACC-Sigma-Aldrich; and Kelly cells (neuroblastoma) were a kind gift from Dr. C. Einvik. HCT-116 and MCF7 cells are WT for TP53 (http://p53.iarc.fr/CellLines.aspx). All cells were grown in DMEM (Life Technologies), except Kelly and PC9 cells, grown in RPMI medium (Life Technologies), both supplemented with 10% fetal bovine serum (Life Technologies) and 0.6% penicillin/streptomycin (Life Technologies). Cells were transfected with a Nucleofector II device (Lonza) using the Amaxa Nucleofector kit (Lonza) and electroporation program recommended by the manufacturer. 293T cells were transfected using polyethylenimine (Polysciences). The efficiency of each transfection was assessed in parallel using a GFP-containing plasmid.
Nutlin 3 was purchased from SelleckChem, and doxorubicin was purchased from Abcam. Gefitinib, WZ4002, TAE684, trametinib, and KU-55933 were purchased from Santa Cruz Biotechnology.

Guernet A., Mungamuri S.K., Cartier D., Sachidanandam R., Jayaprakash A., Adriouch S., Vezain M., Charbonnier F., Rohkin G., Coutant S., Yao S., Ainani H., Alexandre D., Tournier I., Boyer O., Aaronson S.A., Anouar Y, & Grumolato L. (2016). CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations. Molecular cell, 63(3), 526-538.

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Evaluation of Anti-Metastatic Drug Compounds

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Animal procedures had approval of the Vanderbilt University Institutional Animal Care and Use Committee. For in vivo metastatic models, NOD-SCID-IL2Rγ_c (NSG) mice (6–8 weeks) (Jackson Laboratory) were injected in the left cardiac ventricle with 1×10⁵ cells/100 μL PBS. Mice injected with MCF-7 cells received a 17β-estradiol pellet (0.36-mg 60-day release; Innovative Research of America). Mice bearing MCF-7 metastasis were injected IP with vehicle (10% (2-hydroxypropyl)-β-cyclodextrin (HPBCD) in 10% DMSO) or (1b) (40 mg/kg) 2h prior to euthanasia (day 50). Mice injected with HDQ-P1-Luc were randomized and at 2 h after injection were treated for 5d with HPBCD, (1b) (40 mg/kg) IP Q12h or trametinib (2 mg/kg, Santa Cruz Biotechnology, Inc) IP Q24 h. For bioluminescence imaging, mice were injected IP with RediJect D-Luciferin (1.5 mg) (PerkinElmer, Inc.) and imaged with a Xenogen IVIS using Living Image acquisition software (Xenogen Corp.). For ex vivo imaging, organs were placed in D-Luciferin (150 mg/mL PBS). After imaging tissue was fixed in 4% buffered formalin and paraffin-embedded.

Ludwik K.A., Campbell J.P., Li M., Li Y., Sandusky Z.M., Pasic L., Sowder M.E., Brenin D.R., Pietenpol J.A., O’Doherty G.A, & Lannigan D.A. (2016). Development of a RSK Inhibitor as a Novel Therapy for Triple Negative Breast Cancer. Molecular cancer therapeutics, 15(11), 2598-2608.

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Haemocyte Primary Culture and Live Imaging

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Primary culture of haemocytes was performed by bleeding ten larvae expressing tub-miniCic::mCherry that were first washed in ethanol, dried on a piece of paper and resuspended in 200 μL of S2 medium on a square of parafilm. After bleeding with forceps each larvae, the 200 μL were deposed in a MaTek Petri dish (P35G-1.5-10-C) and we let the cells sediment and adhere for 30min. The media was then supplemented by 2mL of S2 medium. The dish was placed on a LSM800 point scanning confocal (63X oil N.A. 1.3) and acquisition was launched with a transmitted light channel and mCherry channel (one z stack/min). Trametinib (Santa Cruz, 10mM stock in DMSO, final concentration 10 μM) or DSMO (1/1000) were added in the medium at the onset of the movie. Nuclear miniCic signal was measured by tracking manually the nucleus using the transmission light channel on Fiji, measuring mCherry intensity and normalizing intensity by intensity at t0.

Moreno E., Valon L., Levillayer F, & Levayer R. (2019). Competition for Space Induces Cell Elimination through Compaction-Driven ERK Downregulation. Current Biology, 29(1), 23-34.e8.

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Gellan Gum-Based Media Preparation and Growth Factor Sourcing

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Gellan gum was purchased from Sansho (Osaka, Japan). In order to prepare gellan gum (FP001) containing media, gellan gum was suspended in pure water to 0.3% (w/v) and dissolved by stirring at 90°C.14 The aqueous solution was sterilized at 121°C for 20 min in an autoclave. The solution was then added to each medium at the given concentration with stirring at room temperature.
Human growth factors, epidermal growth factor (EGF), heparin‐binding EGF‐like growth factor (HB‐EGF), transforming growth factor‐β (TGF‐β), basic fibroblast growth factor, insulin‐like growth factor 1, and platelet‐derived growth factor‐BB, were purchased from PeproTech (Rocky Hill, NJ, USA). Gefitinib, erlotinib, trametinib, and MK2206 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Adriamycin, paclitaxel, and mitomycin C were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Aihara A., Abe N., Saruhashi K., Kanaki T, & Nishino T. (2016). Novel 3‐D cell culture system for in vitro evaluation of anticancer drugs under anchorage‐independent conditions. Cancer Science, 107(12), 1858-1866.

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