Derivatization of polyvinylidene difluoride membranes for the solid-phase sequence analysis of a phosphorylated sea urchin embryo histone H1 peptide.
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.
Rodrigues
J D
JD
Department of Biochemistry, FRD-UCT Research Centre for Molecular Biology, University of Cape Town, Rondebosch, Republic of South Africa.
Combrink
J
J
Brandt
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D006657:Histones / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011145:Polyvinyls; D012617:Sea Urchins / Q000196:embryology; D017421:Sequence Analysis / Q000379:methods*; D005465:Fluorocarbon Polymers
D006657:Histones; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers