= Ligand-dependent membrane phosphorylation experiment
A motivating experiment used isolated membranes from A-431 <biological cells>, which supplied abundant <EGFR>. Separate membrane aliquots were incubated at $0\,{}^{\circ}\mathrm C$ with <ATP> carrying radioactive <phosphorus> in its terminal <phosphate> group, plus <magnesium> or <manganese> <ions>. Radioactivity in material recovered by <chemical acid>-induced <precipitation (chemistry)> supplied the readout. Adding <EGF> beforehand enhanced incorporation relative to the unstimulated preparation; this placed a ligand-dependent biochemical response in a cell-free membrane system.<Epidermal Growth Factor by Stanley Cohen>[Cohen, Epidermal Growth Factor, printed p. 339: A-431 membrane aliquots, terminally labeled ATP, divalent ions and trichloroacetic acid-insoluble incorporation]{ref}
The follow-up comparisons separated increased addition from suppressed removal: <phosphate> removal was rapid but unaffected by <EGF>, and anti-<EGF> <antibody> reversed activation by removing the <ligand>. These observations support reversible regulation of membrane-associated <protein kinase> activity.<Epidermal Growth Factor by Stanley Cohen>[printed p. 341, numbered observations 1–4: rapid activation, dephosphorylation, lack of soluble kinase release and anti-EGF reversal]{ref} Total incorporation alone cannot identify which <protein> or <amino acid residue> received the label, prove intrinsic receptor <catalysis>, or predict an intact <biological cell>'s response.
The same account records an instructive identification error: receptor <phosphorylation> was initially assigned to the wrong <amino acid residue> because released modified <amino acids> migrated together in the electrophoretic <electric field>. Reanalysis identified <phosphotyrosine residues>. Retained radioactivity is evidence about the assay's separation and detection, not unique chemical identity.<Epidermal Growth Factor by Stanley Cohen>[printed p. 341: co-migration in the electrophoretic system and subsequent identification of tyrosine phosphorylation]{ref}
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