Difference between revisions of "Team:Freiburg/Results/Surface"
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| + | Using a cell-free expression system requires the establishment of a specific surface that prevents the binding of non-target proteins. A stable protocol for the production of Ni-NTA surfaces was developed. The 10xHis Tag complexes nickel ions on the surface resulting in a sufficiently strong binding of the protein. In figure 4, an unspecific PDITC surface and a Ni-NTA surface are compared. Flushing the slides with the same antibody solution resulted in a weaker signal for PDITC, which is explained by the binding of many non-target proteins blocking the surface for the His-tagged protein. | ||
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| + | Additionally, we have shown that cell-free expressed and therefore non-purified protein can be efficiently immobilized on the surface in a sufficient amount to detect antibody binding by iRIf (figure 5). | ||
| + | Figure 6 shows that several different antigen spots can be distinguished by flushing the slide with antibodies that are specifically binding to just one of them. Only the spot where the antigen corresponding to the used antibody is immobilized exhibits a signal. | ||
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| + | The establishment of such a specific and reliable surface chemistry was a really challenging task. <a href="https://2015.igem.org/Team:Freiburg/Results/Surface"> Here, you find all the improvements we achieved in this field.</a> | ||
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| + | <div class="thumb2 trien" style="width:310px"><div class="thumbinner">Invalid Link<div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&media=figure_name2.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 4: Quotion picture of an iRIf meassurement of non-purified, His-tagged GFP(?) on PDITC compared to Ni-NTA.</strong> Whole cell lysate was spotted either on an unspecific PDITC surface (A) or on a specific Ni-NTA surface (B) and flushed with anti-GFP(?). The quotion picture shows the change in thickness at distinct spots related to the rest of the surface.</div></div></div><div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/6/6d/Freiburg_2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&media=2015_freiburg_cellfex_gfp_on_ni-nta_binding_curve.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 5: Binding curve of anti-GFP to cell-free expressed GFP on a Ni-NTA surface.</strong> Cell-free expressed GFP was spotted on a specific Ni-NTA surface and flushed with anti-GFP. The binding curve indicated a binding event a certain spot.</div></div></div> | ||
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<h2 class="sectionedit1">Results: binding on surface</h2> | <h2 class="sectionedit1">Results: binding on surface</h2> | ||
Revision as of 21:25, 12 September 2015
Using a cell-free expression system requires the establishment of a specific surface that prevents the binding of non-target proteins. A stable protocol for the production of Ni-NTA surfaces was developed. The 10xHis Tag complexes nickel ions on the surface resulting in a sufficiently strong binding of the protein. In figure 4, an unspecific PDITC surface and a Ni-NTA surface are compared. Flushing the slides with the same antibody solution resulted in a weaker signal for PDITC, which is explained by the binding of many non-target proteins blocking the surface for the His-tagged protein.
Additionally, we have shown that cell-free expressed and therefore non-purified protein can be efficiently immobilized on the surface in a sufficient amount to detect antibody binding by iRIf (figure 5).
Figure 6 shows that several different antigen spots can be distinguished by flushing the slide with antibodies that are specifically binding to just one of them. Only the spot where the antigen corresponding to the used antibody is immobilized exhibits a signal.
The establishment of such a specific and reliable surface chemistry was a really challenging task. Here, you find all the improvements we achieved in this field.
Results: binding on surface
One of the crucial parts of our system is the specific binding of antigens to our glass slide, which is mediated by a multilayer surface chemistry. In our final setup the antigens, which are geneticly fused to a tag, are expressed cell free. In order to produce proteins without the involvment of cells, a complex mixture of various proteins and chemical additives is needed. To prevent binding of These proteins from the cell free transcription and translation mix and simultaneously bind tagged antigens we need a specific surface chemistry. We built up our own surfaces from scratch, starting with two silanes that are bound covalently to the glass slide after it was activated with oxygen plasma. The silane GOPTS (3-Glycidyloxypropyltrimethoxysilane) has an epoxy group that can bind to amino groups, hydroxy or thiole Groups (see surchem methods). The silane APTES (3-Aminopropyltriethoxysilane) has an amino group that can be linked to other amino groups through PDITC (p-Phenyldiisothiocyanate).(see surchem methods)
We compared the binding capacity of these two functionalized surfaces to decide, which one is better suited to function as basis for a specific surface. Therefore we measured the fluorescence intensity of immobilized GFP of different concentrations. As the graph shows especially for low concentration the APTES/PDITC surface bound more GFP. Which is why we chose to continue with APTES/PDITC.
As soon as our first surface was ready we tested it in iRIf and tried to detect the interaction of an anti-GFP antibody to spotted GFP of different concentrations. We got three purified GFPs from different groups and tried all of them to see, which one is working best for us. We spotted the GFPs according to the following pattern:
| GFP 1, 50 µg/ml | GFP 1, 17 µg/ml | GFP 1, 5 µg/ml |
| GFP 2, 50 µg/ml | GFP 2, 17 µg/ml | GFP 2, 5 µg/ml |
| GFP 3, 50 µg/ml | GFP 3, 17 µg/ml | GFP 3 ,5 µg/ml |
| bBSA (0.5 mg/mL) | BSA (10 mg/mL) |
The quotient pictures show binding of anti-GFP antibodies to the immobilized GFP for each concentration. When Cy5 labeled streptavidin (Strep-Cy5) was flushed over the slide not only the spot with bBSA (biotinylated bovine serum albumin), which functions as positive control, lightens up but also the signal of all the GFP spots increases. As the used GFP antibody is biotinylated, this was expected and confirms that the anti-GFP is bound on the surface.
On the foundation of our unspecific surface we constructed a Ni-NTA (Nickel-Nitrolotriacetic acid) surface that should bind our 10x-His-tagged antigens specifically. To determine the specificity of this Ni-NTA surface we compared it to the PDITC surface. We spotted His-GFP-lysate and untagged GFP-lysate as well as purified His-GFP on the surfaces and measured the fluorescence intensity. The graph below shows the mean fluorescence intensity for all spots. The intensity for the His-GFP lysate on the Ni-NTA surface is 4 times higher than on the PDITC surface, while the values for the purified His-GFP are nearly the same. The mean intensity for the untagged GFP-lysate is in the range of the background for the Ni-NTA surface and just slightly higher for the PDITC surface, which shows, that the GFP cannot bind to Ni-NTA without a His-tag. The low intensity for the lysates on PDITC indicates, that all lysate proteins including the GFP are bound to the surface.
After we validated the specificity of our Ni-NTA surface and were able to successfully express GFP with our self-established cell free expression mix (link to cellfree), we tested if the amount of cell free expressed GFP bound by the Ni-NTA surface was high enough to be detectable in iRIf. Therefore we designed a experiment in which we spotted differently expressed and purified GFPs on a glass slide and flushed it with anti GFP antibodies.
| # | Spot |
|---|---|
| 1-3 | Cell free expressed His-GFP |
| 4-6 | Cell free expression mix without DNA (neg) |
| 7-8 | His-GFP lysate |
| 9 | Untagged GFP lysate (unspecific binding) |
| 10 | bBSA (unspecific binding) |
| 11 | Purified GFP (1 mg/mL) |
The strong signal for the spots 1-3 indicates the binding of anti GFP antibodies to the cell free expressed GFP. This means enough GFP molecules are immobilized directly from the cell free expression mix by our Ni-NTA surface to be detectable with iRIf. As expected, the His-GFP lysate spots(7-8) showed a strong signal, while for the untagged GFP lysate (spot 9) and bBSA (spot 10) almost no signal was detected. The purified GFP (spot 11) caused a high signal, but is only partially visible due to the formation of an air bubble on its location. By demonstrating that our surface is able not only to specificly immobilize tagged proteins, but also in sufficient amounts we are one major step closer to a fully functional DIAchip.
While searching for the surface that fits best to our needs we also tried out the Halo-Tag-Ligand system. The Halo-Tag binds covalently to chloralkanes which are immobilized on the surface. We tested several different ligands, which differed in length of the alkane chain and surface attachment method. The ligand that worked best for us in the end, was a 3-chloropropylsilane, which we directly immobilized on plasma activated iRIf slides. To test the surface we spotted our self-expressed Halo-GFP and Halo-mCherry as well as purified Halo-GFP as a positive control and purified untagged GFP as a negative control, which we both got from a research group from Osnabrück. As an additional negative control we spotted bBSA.
The iRIf measurement showed that the Halo-tagged GFPs were successfully immobilized on the surface. Unfortunately there was a lot of unspecific binding, so that the negative control nearly bound as much to the surface as the positive control. The needed optimizations that would be necessary for a specific surface could not be performed due to time limitations. We decided to work with the Ni-NTA surface we established for future experiments.
