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

Use a total-protein stain or validated constitutive loading control, and measure total amounts of the candidate signaling proteins alongside their site-specific phosphorylated forms. This distinguishes increased phosphorylation fraction from increased protein abundance. Include a no-primary or secondary-only control and a phosphatase-treated aliquot to test phospho-dependent detection. Confirm candidate identities using validated target-specific phospho-antibodies, suitable positive controls and, where feasible, depletion or inhibition of the relevant target. Repeat the paired time course with biological replicates and non-saturated imaging. Matching culture state, stimulus dose and sampling time is essential because an ordinary difference in response kinetics could otherwise mimic constitutive activation.