Amino acid analysis on polyvinylidene difluoride membranes.
A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.
Tous
G I
GI
Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854.
Fausnaugh
J L
JL
Akinyosoye
O
O
Lackland
H
H
Winter-Cash
P
P
Vitorica
F J
FJ
Stein
S
S
D000596:Amino Acids / Q000032:analysis*; D002256:Carbonic Anhydrases / Q000032:analysis; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008567:Membranes, Artificial; D011145:Polyvinyls; D012394:Rosaniline Dyes; D012709:Serum Albumin / Q000032:analysis; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D002256:Carbonic Anhydrases; D008567:Membranes, Artificial; D011145:Polyvinyls; D012394:Rosaniline Dyes; D012709:Serum Albumin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C004692:coomassie Brilliant Blue