Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis.
We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.
Shimazaki
Youji
Y
Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577, Japan. yoji@dpc.ehime-u.ac.jp
Kadota
Mariko
M
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D051379:Mice; D010088:Oxidoreductases / Q000032:analysis / Q000737:chemistry*; D011145:Polyvinyls / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D005465:Fluorocarbon Polymers
D004800:Enzymes, Immobilized; D010088:Oxidoreductases; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers