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    Characterization and Immunodetection of Human Glucose-6-Phosphate Dehydrogenase

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    Author
    Le, Karen
    Issue
    urc_student; urc_student
    Date
    2003-01-01 0:00
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    URI
    https://scholar.oxy.edu/handle/20.500.12711/285
    Abstract
    In the process of gel electrophoresis and Western blotting, human glucose-6-phosphate dehydrogenase (G6PD) is separated from other proteins, transferred to a nitrocellulose membrane and further detected by the use of antibodies. The task of this research was to assign a quantitative molecular weight range to G6PD bands observed in SDS and native gels and to revise the protocol of Western blotting of the enzyme. In the SDS polyacrylamide gel electrophoresis (SDS-PAGE), G6PD existed primarily as a monomer (MW ~59 kDa). In native PAGE, the protein was not denatured, and retained some of its biological activity and interactions. The results showed that G6PD in old samples, tended to aggregate to become octamers (MW ~480 kDa). In samples purified this year, the tetramers (MW 236 kDa) seemed to be in greater concentration. After electrophoresis, G6PD on the gel was transferred to a nitrocellulose membrane. The membrane was blocked from nonspecific protein binding sites in a blocking buffer (5% milk, 0.1% Tween 20 in TBS. Next, it was incubated in the anti human G6PD- (rabbit polyclonal) solution at a concentration of 0.4 g/ml, and in a 0.06 g/ml solution of anti Rabbit IgG, Alkaline Phosphate (AP) conjugate. The final step was to develop the membrane with the BCIP/NBT solution, which reacted with the AP to give a purple precipitation. The membranes from both SDS and native gels resulted in distinct, purple bands.
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