Electroblotting of polypeptides onto glass fiber filters for direct sequence analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The technique of electroblotting polypeptides onto Polybrene-treated glass fiber filter discs after protein detection with potassium chloride is evaluated further with different proteins in separate applications. The number of proteins analyzed with this method is now more than double that previously reported. Reproducible results in good yield are obtained. Average overall yield--including the electrophoretic step before blotting--is 26%, with maximal recoveries through all steps up to 60%. High sensitivity radiosequence analysis is also applicable. Recent modifications of the previously described procedure include use of Whatman glass fiber filters, removal of air in the Polybrene-impregnated filters by buffer penetration under reduced pressure, and use of widely different times for electrotransfer. Special advantages with this method are low extent of protein alpha-amino group destruction, direct use of the entire filter in the sequencer, and insensitivity to variations in electroblotting time. Gas-phase hydrolysis in situ of blotted proteins followed by amino acid analysis is known to give a low yield of polar amino acids, and often artifacts, but can still give an estimate of the amount of polypeptide immobilized on the filter. A wash with n-butyl chloride is now shown to reduce the Polybrene-associated artifacts, and an addition of sodium chloride to increase the recovery of polar amino acids. These two steps therefore appear interesting in schemes for compositional analyses of electroblotted proteins.
Bergman
T
T
Department of Chemistry I, Karolinska Institutet, Stockholm, Sweden.
Jörnvall
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005898:Glass; D006583:Hexadimethrine Bromide; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011506:Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate
D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D006583:Hexadimethrine Bromide