Detection of anti-GFP antibodies in rabbit serum using cell-free expressed GFP

Figure 1: Binding of anti-GFP antibodies to cell-free expressed GFP. Cell-free expressed GFP was spotted on a specific Ni-NTA slide and flushed with serum of a rabbit immunized against GFP. The lowest spot consisting of cell-free expressed GFP shows a significant signal compared to the negative control (middle spot).

hier noch rein bringen, dass ihr euren eigenen cell-free mix hergestellt habt und mit diesem auch GFP expremieren könnt und es im array erfolgreich nachweisen könnt

To show that cell-free expressed proteins can be immobilized on the protein surface while maintaining their antibody binding properties, the expression mix was spotted on a specific Ni-NTA surface after GFP expression.
Serum of a rabbit immunized with GFP was applied to the DiaCHIP, which resulted in a strong signal at the referring spot evoked by antibody binding events (figure 1). Thus, His-tagged GFP was successfully expressed and immobilized on the surface, still enabling anti-GFP antibodies in a serum sample to bind.
Find out more about the preparation of the DiaCHIP by producing a protein microarray from a DNA template using cell-free expression.

Building our very own, low-cost DiaCHIP measuring device.

Figure 2: Functional, but low-cost variant of the measuring device. Our self-built device consistent of not much more than two lenses and a camera (A) reliably detected the binding of anti-GFP to GFP (B).

The measuring device we used in collaboration with the AG Roth (ZBSA) to detect antibody binding events is a really expensive machine, but the physics which it is based on is rather simple. To enable future iGEM teams to profit from this label-free detection method as we did, a cost-efficient and easy to rebuild variant of the device was developed (figure 2A). Figure 2B shows the binding of anti-GFP to GFP as it was established before, measured with our own device.
Look here, to see how you can build your own device for label-free detection of antibody binding on a protein microarray needing two lenses, a camera and a little bit of fine feeling (??) sensitivety/sensitiveness/finesse/sure instincts.