HEK 293T cells were transiently transfected in 10-cm plates with cmyc-R11.1.6, cmyc-YW1, HA-K-Ras G12D, HA-K-Ras WT, or a combination thereof as indicated. Approximately 24 hours after transfection, cells were lysed in protease inhibitor (cOmplete EDTA-free protease inhibitor cocktail, Roche) containing NP-40 lysis buffer (Abcam). Whole cell lysates were analyzed by western blot for activation of MEK, ERK, and AKT with phosphospecific antibodies. Experiment was performed in triplicate. pMEK, MEK, pERK, and ERK bands were quantified using ImageJ software.
Np 40 lysis buffer
Manufactured by Abcam
Sourced in United Kingdom
NP-40 lysis buffer is a non-ionic detergent-based solution designed for the extraction and solubilization of proteins from cells and tissues. It is commonly used in a variety of biochemical applications, such as protein purification, immunoprecipitation, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Diphenylamine, by Merck Group (1 mentions)
Non fat dry milk, by Merck Group (1 mentions)
Jc 10, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Dichloro dihydro fluorescein diacetate dcfh da, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Anti tubulin, by Abcam (1 mentions)
Anti ha beads, by Thermo Fisher Scientific (1 mentions)
Anti c myc beads, by Thermo Fisher Scientific (1 mentions)
4 protocols using np 40 lysis buffer
1
HEK 293T Transfection and Signaling
2
Anticancer Potential of Venom Across Breast Cell Lines
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For this exprimental in vitro study, two breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human mammary epithelial cell line (MCF-10a) were obtained from the National Cell Bank of Iran. Trypsin, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), acridine orange (AO), ethidium bromide (EB), dimethyl sulfoxide (DMSO), tween-20, propidium iodide (PI), JC-10, dichloro- dihydro-fluorescein diacetate (DCFH-DA), non-fat dry milk, and diphenylamine were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). NP-40 lysis buffer, antibodies, and enhanced chemiluminescence (ECL) western blotting substrate were purchased from AbCam (Cambridge, UK). Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were procured from Gibco (USA). Lyophilized venom was supplied by Razi Vaccine and Serum Research Institute. Cells were treated with 0, 0.62, 1.25, 2.5, 5, and 10 μg/mL of venom in cell culture media.
3
Western Blot Analysis of CCT7 Protein
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Proteins were isolated from EC tissues via NP-40 lysis buffer (Abcam Ltd., Cambridge, UK) and then separated in 12% SDS-PAGE gels and blotted on nitrocellulose membranes. The filters were hybridized with polyclonal anti-CCT7 (Abcam Ltd., Cambridge, UK) at 4°C overnight, followed by incubation with the secondary anti-rabbit (Abcam Ltd., Cambridge, UK) for 1 h at room temperature. Anti-Tubulin (Abcam Ltd., Cambridge, UK) was used as the loading control. Gray-scale values were analyzed using the ImageJ software and were also described previously (19 (link)).
4
Investigating K-Ras G12D-B-Raf Interactions
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HEK 293T cells were transiently transfected in 10-cm plates with cmyc-R11.1.6, cmyc-YW1, HA-K-Ras G12D, or a combination thereof as indicated. Approximately 24 hours after transfection, cells were lysed in protease inhibitor (cOmplete EDTA-free protease inhibitor cocktail, Roche) containing NP-40 lysis buffer (Abcam). Whole cell lysates were analyzed by western blot for total HA-K-Ras G12D and B-Raf. Cmyc-tagged R11.1.6/YW1 were pulled down with anti-cmyc beads (Thermo Scientific) and analyzed for co-precipitation of HA-K-Ras G12D by western blotting and co-precipitation of other intracellular proteins by SDS-PAGE and silver stain (Thermo Scientific). Experiment was performed in duplicate. HA-tagged K-Ras G12D was pulled down with anti-HA beads (Thermo Scientific) and analyzed for co-precipitation of endogenous B-Raf by western blotting. Experiment was performed in triplicate.
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