Team:Freiburg/Results

From 2011.igem.org

(Difference between revisions)
(Results)
(Plastic binding domain)
Line 208: Line 208:
===<span style="color:grey;">Plastic binding domain</span>===
===<span style="color:grey;">Plastic binding domain</span>===
-
[[Image:picture 1 pbd.jpg|300px|left|thumb|Picture 1: % of tagged (red) or untagged (blue) GFP remaining in the well after washing compared to previous washing step / original concentration]] After the first measurement of the basic fluorescence intensity we transferred the samples onto another well, refilled the exhausted well with PBS and measured both eluate and remaining protein. In a second washing step the liquid was taken out of the first well again and given to another well. This washing was performed a third time, resulting in three eluates and a triply washed well with more or less protein remaining on the walls.
+
[[Image:picture 1 pbd.jpg|300px|right|thumb|Picture 1: % of tagged (red) or untagged (blue) GFP remaining in the well after washing compared to previous washing step / original concentration]] After the first measurement of the basic fluorescence intensity we transferred the samples onto another well, refilled the exhausted well with PBS and measured both eluate and remaining protein. In a second washing step the liquid was taken out of the first well again and given to another well. This washing was performed a third time, resulting in three eluates and a triply washed well with more or less protein remaining on the walls.
-
As shown in picture 1, both pbd-tagged and untagged GFP fluorescence decreases after the first washing step. Only 0- 4% of the original concentrations of the untagged GFP and 2-7% of the pbd-tagged GFP remained in the well. After a second washing step there were differences observable between the tagged and the untagged GFP.  While the percentage of the remaining “normal” GFP was scattering around zero, averagely 60% of the pbd-tagged GFP remained in the well. In a third washing steps this observation was confirmed. While again the main part of the pbd-GFP remained in its original well there could rarely been found any untagged GFP that hadn’t been washed away.
+
As shown in picture 1, both pbd-tagged and untagged GFP fluorescence decreases after the first washing step. Only 0- 4% of the original concentrations of the untagged GFP and 2-7% of the pbd-tagged GFP remained in the well. After a second washing step there were differences observable between the tagged and the untagged GFP.  While the percentage of the remaining “normal” GFP was scattering around zero, averagely 60% of the pbd-tagged GFP remained in the well. In a third washing steps this observation was confirmed. While again the main part of the pbd-GFP remained in its original well there could rarely been found any untagged GFP that hadn’t been washed away.[[Image:picture 2 pbd.jpg|300px|right|thumb|Picture 2: % of pbd-tagged GFP that can be eluted in the first washing step. The curve decreases with the amount of the start concentration. If the GFP-pbd solution is not oversaturated a lower amount of this protein can be washed away.]]
-
In case of the pbd-bound GFP it is striking that after the solution has already been diluted by one washing step, the percentage of protein that can be washed away diminishes. It comes to mind that the massive lost of pbd-tagged GFP after the first washing step might be due to an oversaturation of pbd-GFP in the solution. [[Image:picture 2 pbd.jpg|300px|left|thumb|Picture 2: % of pbd-tagged GFP that can be eluted in the first washing step. The curve decreases with the amount of the start concentration. If the GFP-pbd solution is not oversaturated a lower amount of this protein can be washed away.]] In this case there would be much more pbd-GFP than place on the plastic surface so that most of the protein could be eluated.
+
In case of the pbd-bound GFP it is striking that after the solution has already been diluted by one washing step, the percentage of protein that can be washed away diminishes. It comes to mind that the massive lost of pbd-tagged GFP after the first washing step might be due to an oversaturation of pbd-GFP in the solution. In this case there would be much more pbd-GFP than place on the plastic surface so that most of the protein could be eluated.
As shown in picture 2 the percentage of eluted GFP diminishes when the used start concentration is lower. As the used [[Image: pbd_better_than_GFP.jpg|300px|right| thumb | Picture 3: if the start concentration is not oversaturated pbd-coupled is much more resistant to washing steps than GFP alone.]]  
As shown in picture 2 the percentage of eluted GFP diminishes when the used start concentration is lower. As the used [[Image: pbd_better_than_GFP.jpg|300px|right| thumb | Picture 3: if the start concentration is not oversaturated pbd-coupled is much more resistant to washing steps than GFP alone.]]  
To investigate this phenomenon  we compared different start concentrations of pbd-GFP concerning the amount of pbd-GFP that could be washed away.
To investigate this phenomenon  we compared different start concentrations of pbd-GFP concerning the amount of pbd-GFP that could be washed away.

Revision as of 01:43, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!