Native electrophoretic separation and femtomolar detection of 65Zn-containing proteins by storage phosphorimaging.
Over 300 zinc-containing proteins have been described and over 1000 genes in the human genome encode proteins with zinc finger domains. Despite the important role of zinc in mammalian physiology, understanding its cellular homeostasis remains limited. In this study, we demonstrate the utility of radioactive zinc detection using nondenaturating, native electrophoresis. We tested 65Zn-labeled enterocyte and enterocyte brush border membrane proteins and 65Zn-metallothionein. The radioactive decay of 65Zn is efficiently captured by storage phosphorimaging screen, enabling detection of 65Zn-labeled metalloproteins in subpicogram range. This powerful technique has a wide potential for studies of zinc absorption, incorporation of zinc into apoproteins and transcription factors that require zinc, zinc turnover in metallothionein, and other aspects of cellular zinc metabolism.
Vyoral
Daniel
D
Institute of Hematology and Blood Transfusion, U nemocnice 1, 2, 128 20 Prague 2, Czech Republic. vyoral@uhkt.cz
Babusiak
Marek
M
Fuchs
Ota
O
Petrák
Jirí
J
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D002478:Cells, Cultured; D004586:Electrophoresis / Q000379:methods*; D020895:Enterocytes / Q000000981:diagnostic imaging / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D008668:Metallothionein / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008832:Microchemistry / Q000379:methods; D008871:Microvilli / Q000000981:diagnostic imaging / Q000378:metabolism*; D011877:Radionuclide Imaging; D016335:Zinc Fingers; D015037:Zinc Radioisotopes
D015037:Zinc Radioisotopes; D008668:Metallothionein