CE-LIF of Green Fluorescent Fusion Proteins for Investigation of Phosphorylation

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Authors

MICHALCOVÁ Lenka DOPITOVÁ Radka HEJÁTKO Jan RYVOLOVÁ Markéta VÁLKOVÁ Martina PREISLER Jan

Year of publication 2011
Type Conference abstract
MU Faculty or unit

Central European Institute of Technology

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Description In this study, CE-LIF with 405 nm laser diode was used to study AHP5 fused with GFP. AHP5-GFP fusion proteins overexpressed in E. coli were used for initial optimization of CE-LIF technique because of lower concentrations of the fusion protein produced by plant. The stability of the AHP5-GFP sample was examined; the complex was found to be unstable with GFP being released. The GFP release was not affected by either a long-term storage at -80 and -20 C or the addition of one of the inhibitors (EDTA, PMSF). These results were confirmed by the western blotting. The phosphorylation of the sample AHP5-GFP in vitro is under investigation. Gel filtration was used to obtain mass fractions for better understanding electrophoregrams. The mass fractions were investigated by CE-LIF and Western blotting-LIF and Western blotting.
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