Direct analysis of retinal dehydrogenase activity on an electroblotting membrane following separation by non-denaturing two-dimensional electrophoresis.
The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes.
Shimazaki
Youji
Y
Graduate school of Science and Engineering (Science section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Kuroda
Takahiro
T
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008567:Membranes, Artificial; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D008567:Membranes, Artificial; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase