Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3.
The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.
Ploug
M
M
Institute of Biochemical Genetics, University of Copenhagen, Denmark.
Jensen
A L
AL
Barkholt
V
V
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003101:Collodion; D003176:Complement C3 / Q000032:analysis; D004563:Electrochemistry / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005998:Glycine / Q000031:analogs & derivatives; D008567:Membranes, Artificial; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000032:analysis*; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D003101:Collodion; D003176:Complement C3; D005998:Glycine; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C100184:tricine