= Solution
Culture each patient and control sample under matched conditions, including equivalent growth-factor withdrawal where appropriate, then prepare paired unstimulated and <PDGF>-stimulated samples with the same stimulation time. Rapidly lyse on ice with suitable detergents and <protease> and <phosphatase> inhibitors. Measure <protein> concentration and load equal amounts after <SDS-PAGE> sample preparation. Separate the <proteins> with molecular-mass markers, transfer them to a suitable membrane, verify transfer and block non-specific binding. Incubate with the Ras-PST25 <primary antibody>, wash, add a detectable <secondary antibody>, wash again and record the signal within its linear range. \b[Preserve the original <phosphorylation> state and compare equal, verifiably transferred inputs.] Include a total-<protein> loading measure, appropriate <antibody> controls and independent biological replicates; use matched exposures rather than independently rescaling lanes.
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