Sample centrifugation onto membranes for sequencing

Abstract

This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accomodate large (>10 μg) and small (<10 μg) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography.