Past exam of the biochemistry and Molecular Biology course of the University of Cambridge / 2025 / Paper 2 / Section B / 13 / f / Solution

mTOR has the supplied molecular mass of about $259\mathrm{kDa}$, so optimize the separation and transfer for a much larger protein. Use a suitably low-percentage or gradient SDS-PAGE gel and high-mass markers, and verify efficient transfer with a protocol appropriate for large proteins, such as optimized wet transfer. Probe with a validated phospho-mTOR reagent and measure total mTOR in parallel. The broad Ras-PST25 reagent cannot be assumed to recognize a useful site on mTOR merely because it recognizes other pathway proteins. Include a positive pathway control and a suitable inhibition or phosphatase control; a missing band on the existing blot could reflect poor separation or transfer rather than absent mTOR phosphorylation.