By Thomas Weichhart
When you consider that its discovery, the mammalian objective of rapamycin (mTOR) has been proven to manage many serious molecular methods in eukaryotes similar to metabolism, progress, survival, getting older, synaptic plasticity, reminiscence, and immunity. In mTOR: equipment and Protocols specialist researchers within the box aspect the various tools that are now generic to check mTOR. those comprise equipment and methods used for the examine of the mTOR pathway and power healing functions of mTOR inhibitors similar to, immunosuppressive and anticancer brokers. Written within the hugely profitable equipment in Molecular Biology™ sequence structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and key tips about troubleshooting and averting recognized pitfalls. Authoritative and functional, mTOR: tools and Protocols seeks to assist scientists within the extra research of this crucially very important molecule starting from the research of sign transduction occasions inside a mobilephone to the evaluate of advanced human ailments corresponding to metabolic issues or melanoma.
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Extra resources for mTOR: Methods and Protocols (Methods in Molecular Biology, v821)
Add 7 mL 10× reducing agent and denature samples at 70°C for 10 min. 13. Spin the sample for 5 s at 14,000 rpm and transfer the supernatant to a new microcentrifuge tube. 2 Biochemical and Pharmacological Inhibition of mTOR… 23 14. Run 5 mL of molecular weight markers and samples in a 26-well 3–8% Tris–acetate gels using Tris–acetate SDS running buffer to resolve mTOR, Rictor, and Raptor. His6-AKT and His6S6K1 are separated in a 26-well 4–12% gels using MOPS SDS running buffer. 15. Remove gel from the cassette and soak in the transfer buffer for 5 min on an orbital shaker.
2, steps 14–24) for P-S6K1(T389), P-AKT (S473), and P-AKT (T308). An example of the results produced is shown in Fig. 3a, left panel. 2. Inhibition of Amino Acid-Induced mTOR Activation 1. Plate HEK293 cells in six-well culture dish in growth medium and let cells to reach 70–80% confluence. 2. Very carefully wash the cells once with amino acid-free medium and incubate in 3 mL amino acid-free medium for 2 h. 3. Amino acid-starved cells are pretreated with 3 mL inhibitors (1,000× dilution from the prepared stocks) for 2 h.
10. Transfer cover slips into 12 wells and wash with PBS three times. 11. Dilute secondary antibodies to 1: 1,000. 12. Incubate with secondary antibodies as mentioned at step 9 under the dark but only for 30 min. 13. Wash with PBS three times and wash once with autoclaved water. 14. Put 20 μl (one big drop) of mounting media onto cover glass and put the cover slips. In this step, causing bubbles should be avoided. 15. Incubate slides for overnight under the dark. 16. Apply clear nail polish around the edge of cover slip to prevent cells from being dried.
mTOR: Methods and Protocols (Methods in Molecular Biology, v821) by Thomas Weichhart