= 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. \b[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.
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