title: Sensitive detection of proteins in polyacrylamide gel via isatoic anhydride derivatization: Introduction of a low-cost fluorescent prelabeling procedure.
abstract: Here, we introduce isatoic anhydride as a sensitive and commodious fluorescent prelabel for detection of proteins in one-dimensional polyacrylamide gels. High reactivity of isatoic anhydride with nucleophiles in mild alkaline environments makes it an appropriate tag for labeling of biomolecules. In this study, we show that preelectrophoresis labeling of proteins with isatoic anhydride for few minutes at room temperature allows detection of 2-4 ng of standard proteins, BSA and lysozyme, per band. Proteins were successfully labeled in the presence of a wide range of common biological reagents and in crude cell extract. The labeled proteins have the same electrophoretic migration in comparison to unlabeled proteins; however the application of saturation labeling method results in slight band broadening. Compatibility of the method with downstream processes was assessed by tryptic digestion of labeled proteins and study of peptide mixture using gel electrophoresis which revealed partial digestion of labeled proteins due to lysine modification. The present procedure is sensitive, rapid, and inexpensive and is a promising alternative for current protein staining procedures, where downstream processes are not desired.
authors.affiliation: Neuro-organic Laboratory, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.
authors.forename: Kazem
authors.initials: K
authors.lastname: Asadollahi
authors.affiliation: Neuro-organic Laboratory, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.
authors.forename: Saharnaz
authors.initials: S
authors.lastname: Rafiee
authors.affiliation: Neuro-organic Laboratory, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran. ghriazi@ut.ac.ir.
authors.forename: Gholamhossein
authors.initials: G
authors.lastname: Riazi
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000191:economics / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000191:economics; D057230:Limit of Detection; D010078:Oxazines / Q000737:chemistry* / Q000191:economics; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D000180:Acrylic Resins; D005456:Fluorescent Dyes; D010078:Oxazines; D011506:Proteins; C016680:polyacrylamide gels; C037902:isatoic anhydride
keywords: Electrophoresis; Fluorescent labeling; Isatoic anhydride; Prelabeling; Protein staining