Derivatization of polyvinylidene difluoride membranes for the solid-phase sequence analysis of a phosphorylated sea...

We describe a simple and effective chemical protocol for attaching polyallylamine to polyvinylidene difluoride (PVDF) membranes. The underivatized PVDF discs were first etched in 0.5 M alcoholic KOH for 1.5 min and subsequently reacted with polyallylamine of high molecular weight under alkaline conditions. The covalently attached amino groups were reacted with 1,4-phenylene diisothiocyanate (DITC), thus converting the amino-modified PVDF discs to DITC-functionalized discs. The substitution level of the DITC-functionalized discs was found to be lower than that of the equivalent discs available commercially. Lysine-containing polypeptides were immobilized on these DITC-membrane discs and then subjected to either solid-phase manual Edman degradation or automated gas-phase sequencing. The method was found to be well suited for the sequencing of 32P-labeled phosphopeptides by employing the manual solid-phase technique.