Selective - The Specific Surface

Figure 4: Comparison of specific Ni-NTA with unspecific PDITC surfaces.The red bars show the mean fluorescence intensity for the different GFPs (His-GFP lysate, untagged GFP lysate, purified His-GFP) on PDITC. The blue bars represent the mean fluorescence intensity for the GFPs on Ni-NTA. The grey bar represents the background for both surfaces.

All the results presented above show, that a detection of antigen-antibody interaction is possible with the PDITC surface chemistry. But as we need a specific surface to bind antigens out of cell-free expression mix, we wanted to build up a Ni-NTA (Nickel-Nitrolotriacetic acid) surface and therefore linked all our target proteins to a 10x Histidine tag. We established a protocol based on the unspecific PDITC surface and optimized it, till we the unspecific binding of other proteins from the cell free mix was minimal. To determine the specificity of our Ni-NTA surface we compared it to the PDITC surface. To asses the purifcation properties we used complete E. coli lysate transformed with either a tagged or an untagged GFP-construct. Additionally purified GFP-His as used in previous experiments was spotted and fluorescence intensity was measured. Figure 4 shows the intensities obtained for all spots: the intensity for the His-GFP lysate on the Ni-NTA surface is about four times higher than on the PDITC surface, while the values for the purified GFP-His are in a comparable range. The mean intensity for the untagged GFP-lysate spot 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 fluorescence intensities for GFP lysate on PDITC is due to the fact, that non-fluorescent proteins in the lysate are bound as efficient as GFP. This way the surface is blocked for further GFP molecules and overall fluorescence decreases compared to the specific Ni-NTA surface.

After we showed that we can successfully bind his-tagged proteins to our Ni-NTA surface, we attempted to immobilize our cell-free expressed His-GFP-Lysate on this surface. You can find the results for this crucial part of our project on the main results page.

Covalent - Other Surface Systems

On the way to find the surface best suited for our needs we also worked with the Promega Halo-tag system. The Halo-Tag that can be fused to target proteins binds covalently to chloroalkanes which are immobilized on the surface. We tested several ligands, which differed in length of the alkane chain and surface attachment method. The ligand that worked best for us, was a 3-chloropropylsilane, which we directly immobilized on plasma activated iRIf slides. To test the surface we pipetted 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 the group of J. Piehler 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 controls nearly bound as much to the surface as the positive control. The 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.

Figure 7: Quotient picture for the Halo-surface measurement. Spot 1-4 show the binding of biotinylated anti-GFP to successfully immobilized self purified Halo-GFP. On spot 5 to 8 Halo-mCherry, but as no anti-mCherry was flushed over, no signal can be observed. On Spot 9 the positive control (Halo-GFP from J. Piehler) was pipetted and on spot 10 the negative control (His-GFP) both show a clear binding to anti-GFP. Also the second negative control bBSA (spot 11) shows a signal after Streptavidine was flushed over.

Figure 8: binding curve for the Halo-surface measurement. The binding curve shows the binding of biotinylated anti-GFP to all The GFPs on the slide (with and without Halo-Tag) and the binding of Streptavidine to the bBSA spot and all the GFP spots, because the anti-GFP that was flushed over before is biotinylated.

Validation of our controls

Figure 9: Western Blots of desalted elution of purified GFP protein with His-Tag. (A) To verify the prescence of purified GFP protein with a His -Tag we performed a Western Blot with anti-His HRP Conjugate (1:1000).(B) Western Blot of GFP-His with specific anti-GFP (1:2000) and anti-Goat HRP antibody (1:5000). The expected molecular weight is 28 kDa.

Given that we were using purified GFP-His Tag for evaluation of our self-made surfaces and in further experiments as positive control we validated it by western blot. Therefore we showed the presence of GFP-His Tag with anti-His HRP Conjugate. Additionally we used the specific anti-GFP antibody as for the detection in iRIf.