By Thomas Weichhart (auth.), Thomas Weichhart (eds.)
Since its discovery, the mammalian objective of rapamycin (mTOR) has been proven to manage many serious molecular methods in eukaryotes akin to metabolism, development, survival, getting older, synaptic plasticity, reminiscence, and immunity. In mTOR: tools and Protocols specialist researchers within the box element a few of the equipment that are now established to check mTOR. those comprise tools and methods used for the examine of the mTOR pathway and power healing purposes of mTOR inhibitors resembling, immunosuppressive and anticancer brokers. Written within the hugely winning Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key pointers on troubleshooting and averting identified pitfalls.
Authoritative and functional, mTOR: equipment and Protocols seeks to help scientists within the additional research of this crucially very important molecule starting from the research of sign transduction occasions inside of a cellphone to the evaluate of complicated human ailments resembling metabolic issues or cancer.
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Extra info for mTOR: Methods and Protocols
Antibodies for IHC and IF are mentioned above. 10. Other Chemicals and Reagents 1. Rapamycin. 2. Wortmannin. 3. Complete Mini, EDTA-free. 4. 5 M NaF and stored at room temperature. 5. 100× Sodium pyrophosphate: 1 M and stored at room temperature. 3. Methods Since the mTOR pathway is extremely sensitive to levels of growth factors and nutrients, maintaining cells in fresh media is always recommended as this keeps mTOR more active, especially when compared to cells cultured in a lower energy environment.
13. Dialyze the eluent with dialysis buffer for 3 h. Dialysis buffer is changed every hour. 14. 5, 1, 2, and 4 μg), which can be a concentration reference. 15. Make aliquots and snap-freeze GST-S6K1 solution using liquid nitrogen. 16. Stored at −80°C until use. 2. Purification of GST-S6 from Bacteria 1. Grow GST-S6 transformed bacteria in 12 ml LB with proper antibiotics for overnight. 2. Scale up overnight culture to 250 ml LB and grow bacteria for additional 1 h at 37°C. 3. 2 mM IPTG for 4–5 h at 37°C.
Wash with TBST four times for 60 min. 12. Completely remove TBST and add ECL mix into the container. 13. Expose the blot on X-ray film in the dark room. 14. Usually, the signal of mTOR, Raptor, Rictor appears within 2–5 min. The signal of phospho- or total S6 and 4EBP1 on X-ray film usually appears within 20 s. S6K1, Akt, and the signal of phosphorylated proteins of these appear within 1 or 2 min. An example of the results produced is shown in Fig. 1 (see Note 2). 4. 1. Preparation of GST-S6K1 from Mammalian Cells In order to monitor kinase activity of mTORC1, S6K1 (ribosomal protein S6 kinase 1) and 4EBP1 (4E-binding protein 1) have been used as substrates.
mTOR: Methods and Protocols by Thomas Weichhart (auth.), Thomas Weichhart (eds.)