Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis of luminescent lanthanide chelate-labeled proteins with time-resolved detection.
Proteins labeled with a luminescent lanthanide chelate, {{2,2',2'',2'''-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2':6',2''-terpyridine-6,6''-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu(3+)),were analyzed with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) slab gel electrophoresis and hydroxyethyl cellurose gel capillary electrophoresis with a time-resolved fluorometric detector. The metal ion of the luminescent lanthanide chelate did not dissociate from the ligand during electrophoresis, and the luminescence was retained. On a slab gel, the band of DTBTA-Eu(3+)-labeled streptavidin was apparently less broad than that of AlexaFluor 488-labeled streptavidin. DTBTA-Eu(3+) in SDS-PAGE slab gel is stable, and the gel after electrophoresis can be dried and stored for at least one year without luminescence fading. In capillary gel electrophoresis (CGE), five labeled proteins with different molecular weight were separated, and a good correlation was observed between the molecular weight and the migration time. Although the detection limits of these proteins determined in buffer solutions of the capillary electrophoresis were not better than those reported for CGE with laser-induced-detection, the detection limits of the same proteins in the present CGE system were not significantly deteriorated in serum solutions compared to those in buffer solutions, and the advantage of using time-resolved detection has been shown.
Yamaguchi
Yoshinori
Y
Consolidated Research Institute for Advanced Science and Medical Care (ASMeW), Waseda University, 513 Wasedatsurumaki-cho, Shinjuku, Tokyo 162-0042, Japan.
Hashino
Kimikazu
K
Ito
Masahiro
M
Ikawa
Keisuke
K
Nishioka
Takuya
T
Matsumoto
Kazuko
K
D000178:Acrylamides / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D049408:Luminescent Agents / Q000737:chemistry*; D009942:Organometallic Compounds / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D019809:Streptavidin / Q000032:analysis*; D013997:Time Factors; D001713:Biphenyl Compounds
D000178:Acrylamides; D002614:Chelating Agents; D049408:Luminescent Agents; D009942:Organometallic Compounds; D019809:Streptavidin; C541078:((2,2',2'',2'''-(4'-)(((4,6-dichloro-1,3,5-triazin-2-yl)amino)biphenyl-4-yl)-2,2':6',2''-terpyridine-6,6''-diyl)bis-(methylenenitrilo))tetrakis(acetato)europium(III); D001713:Biphenyl Compounds