Microsequencing of proteins and peptides in the Knauer sequencer with and without covalent attachment to polyvinylidene difluoride membranes by the wet-phase degradation technique.
Proteins and large peptides were degraded with phenylisothiocyanate (PITC) in the horizontal flow-through-reactor of the Modular Knauer Sequencer (Fischer, S., Reimann, F. & Wittmann-Liebold, B. (1989) in Methods in Protein Sequence Analysis (Wittmann-Liebold, B., ed.) Springer-Verlag, Berlin, pp. 98-107) by the wet-phase filter technique (Wittmann-Liebold, B. (1988) J. Prot. Chem. 7, 224-225) employing polyvinylidene difluoride (PVDF) membranes without polybrene. In order to prevent losses of small peptides during solvent washes at the degradation, 1.4-phenylene diisothiocyanate (DITC) derivatized PVDF support (MilliGen, Burlington, MA) was used to covalently attach the peptide via its lysine groups in situ within the cross-flow reaction chamber onto this membrane (Herfurth, E., Pilling, U. & Wittmann-Liebold, B. (1990) J. Prot. Chem. 9, 267). We found these membranes very suitable for peptide degradations in the Knauer sequencer. In almost all cases we were able to identify the amino-acid residues of the peptide up to its last covalent fixation point to the membrane.
Herfurth
E
E
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin.
Pilling
U
U
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; D012259:Ribonuclease, Pancreatic; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers