'In situ' Edman degradation of protein(s) blotted to immobilon membranes suitable for unblocking reversible chemical modification and elimination of coomassie blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Conditions for unblocking reversible chemical modifications such as maleylation or citraconylation 'in situ' at the N-terminus of proteins after transfer of proteins to immobilon membranes from SDS-PAGE are described. Demaleylation or decitraconylation occurred at 55 degrees C in 70% formic acid (pH 1.50) during 60 min. During the unblocking reaction, Coomassie blue dye was completely removed, resulting in superior high performance liquid chromatographic separation of phenylthiohydantoin-amino acid (PTH-AA) after Edman degradation (automatic gas phase sequencer). The protein fixed on the matrix after demaleylation and removal of Coomassie blue was not degraded. The possible cleavage at the aspartyl-prolyl peptide bonds was considered, but no side reaction was observed. Furthermore, the incubation time in 70% formic acid at 55 degrees C could be reduced to 10 min in the absence of maleylation of the starting material, and this was suitable for the removal of Coomassie blue and the quantification of phenylthiolhydantoin-amino acids (PTH-AAs) by HPLC. The yield from the starting protein through SDS-PAGE, blotting, and Edman degradation to quantitative analysis of PTH-aminoacid(s) by HPLC was established.
Chirat
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F
URA No 409 du CNRS, Institut de Recherche sur le Cancer de Lille (IRCL), France.
Belaiche
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D
Malki
N
N
Han
K K
KK
D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D011145:Polyvinyls; D011506:Proteins; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C048139:Coomassie blue