Derivatization of Polyvinylidene Difluoride Membranes for the Solid-Phase Sequence Analysis of a Phosphorylated Sea Urchin Embryo Histone H1 Peptide
✍ Scribed by J.D. Rodrigues; J. Combrink; W.F. Brandt
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 716 KB
- Volume
- 216
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
We describe a simple and effective chemical protocol for attaching polyallylamine to polyvinylidene difluoride (PVDF) membranes. The underivatized PVDF dises were first etched in (0.5 \mathrm{~m}) alcoholic (\mathrm{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 dises 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 DITCmembrane discs and then subjected to either solidphase manual Edman degradation or automated gas-phase sequencing. The method was found to be well suited for the sequencing of ({ }^{32} P)-labeled phosphopeptides by employing the manual solid-phase technique. c 1994 Academic Press. Inc.