Biomod/2011/TUM/TNT/Results/Folding Purification: Difference between revisions

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The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 2. The results of the slower ramps 2D_H3_ML and 5D_H3_ML were not better than the 15_65 ramp.  
The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar results as the 15_65 ramp.  


<center>[[Image:2011.08.24 theU 1kBlad-scaff-bm1-bm2.jpg]]</center>
<center>[[Image:2011.08.24 theU 1kBlad-scaff-bm1-bm2.jpg]]</center>
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Also there was only one major band visible in the agarose gel (figure 1). This means that no significant amount of byproducts had been formed. Most of the structures from the major band were folded correctly, which was also proven by TEM images.
Most of the structures from the major band were folded correctly, which was proven by TEM images (figure 2).


<center>[[Image:20111021controltheUBM2 2B7.png|700x700px]]</center>
<center>[[Image:20111021controltheUBM2 2B7.png|700x700px]]</center>
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The purification of the structures was done with both an agarose gel as well as an Amicon size exclusion filter. The concentrations of the purified origami were approximately 2 nM when using the gel method and therefor low compared to the filtration with ca. 10 nM.  
The purification of the structures was tried both with an agarose gel and with an Amicon size exclusion filter (molecular weight cutoff: 100kDa). According to general experience, the yield of purification via agarose gel is approximately 2 nM. The yield of the filter purification was definitively higher, since unexpected weak dilutions led to appropriate concentrations for TEM and fluorescence microscope. Therefore we estimate the yield to be roughly 10 nM.  




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