The use of fluorescamine as a detection reagent in protein microcharacterization.
With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.
Miedel
M C
MC
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Hulmes
J D
JD
Pan
Y C
YC
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D005450:Fluorescamine; D008969:Molecular Sequence Data; D010191:Pancreatic Polypeptide; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification; D051381:Rats; D013141:Spiro Compounds; D014357:Trypsin
D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D013141:Spiro Compounds; D005450:Fluorescamine; D010191:Pancreatic Polypeptide; D014357:Trypsin