Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and centrifuge blotting: preparation of polypeptides for amino-terminal sequence analysis.
The applicability and reproducibility of a previously described (L. F. Hermansen et al., Electrophoresis 1993, 14, 1302-1306) centrifuge-blotting procedure for capturing subnanomolar amounts of protein on polyvinylidene difluoride membranes for direct Edman degradation was further investigated. Proteins with different molecular masses were centrifuge-blotted onto Immobilon CD membranes. Simultaneous blotting and desalting was achieved with an overall yield of 15-56% after 2 h centrifugation for proteins with a molecular mass of 12-30 kDa. Centrifugation of myoglobin for 6 h resulted in an overall yield of 72%. The subnanomolar amounts obtained were also sufficient to conduct cyanogen bromide cleavage in situ on proteins with blocked NH2-terminus and to generate sequence information.
Hermansen
L F
LF
Department of Biochemistry, University of Oslo, Norway.
Juul
J
J
Sletten
K
K
D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015151:Immunoblotting / Q000379:methods*; D008567:Membranes, Artificial; D010455:Peptides / Q000032:analysis*; D011145:Polyvinyls / Q000737:chemistry*; D017421:Sequence Analysis; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013997:Time Factors; D005465:Fluorocarbon Polymers
D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers