Team:Freiburg/Results

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(Experimental setup)
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===Experimental setup===
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==Experimental setup==
According to ''Adey et al.'' the plastic binding domain (pbd) binds to the polystyrene surface of micro titer plates (96 well plates). To investigate the binding properties of the plastic binding tag we started several spectroscopic assays using a plate reader (FLUOstar Omega) and polystyrene plates (Greiner bio one) To detect the fluorescence of the GFP tagged to the plastic binding domain we used black plates and a well scanning program measuring 10x10 spots in each well of the micro titer plate. We performed several washing steps to find out how much of the proteins can be found in the eluate and how much remains bound on the plate’s surface. To compare the pbd-tagged GFP to a normal GFP without special plastic binding ability we also measured GFP obtained via expression with our diverse PR (Promoter-Ribosome-binding-site constructs). To affirm the results obtained by fluorescence spectroscopy used the Bradford assay and screened for different protein concentrations in transparent polystyrene plates.
According to ''Adey et al.'' the plastic binding domain (pbd) binds to the polystyrene surface of micro titer plates (96 well plates). To investigate the binding properties of the plastic binding tag we started several spectroscopic assays using a plate reader (FLUOstar Omega) and polystyrene plates (Greiner bio one) To detect the fluorescence of the GFP tagged to the plastic binding domain we used black plates and a well scanning program measuring 10x10 spots in each well of the micro titer plate. We performed several washing steps to find out how much of the proteins can be found in the eluate and how much remains bound on the plate’s surface. To compare the pbd-tagged GFP to a normal GFP without special plastic binding ability we also measured GFP obtained via expression with our diverse PR (Promoter-Ribosome-binding-site constructs). To affirm the results obtained by fluorescence spectroscopy used the Bradford assay and screened for different protein concentrations in transparent polystyrene plates.
The calibration lines necessary for calculation of protein concentration can be found [[Media:Bradford+Fluorescence calibration.pdf|here]].
The calibration lines necessary for calculation of protein concentration can be found [[Media:Bradford+Fluorescence calibration.pdf|here]].

Revision as of 02:42, 22 September 2011


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of the Freiburger student
team competing for iGEM 2011.
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