Based on a method proposed by Araújo et al. (Araújo et al., 2012) DNA was immobilized on Whatman Filter paper previously activated wth p-phenylene-diisothiocyanate. To accomplish this, aminolabeled DNA is required. We made some adaptations in order to immobilize dsDNA instead of ssDNA. The original protocol demanded an 0.5 hs washing step with 4x SSC buffer, which we omitted, since this serves for the denaturing of DNA. Furthermore to be able to quantify the DNA immobilized on paper we hybridized the amino labeled operatorstrand with the complementary strand that was Cy3 labeled, this was performed via a simple annealing of two primers. So by detecting the Cy3 label via the Typhoon Fluorescence scanner we could be sure, that the immobilized DNA was the correct dsDNA.
We were not able to acquire molecular sieves at the start of our project, so we dissolved the p-phenylene diisothiocyanate (PDITC) in pure ethanol, which is easily available in any standard molecular biology laboratory. We compared the loss of the Cy3 fluorescence signal upon washing on paper that was activated with PDITC disolved in ethanol to the loss of signal upon washing on paper that was activated with PDITC disolved in dried DMSO. The loss of signal can be mainly attributed to the washing out of DNA, since an control showed, that the decrease of fluorescence of the Cy3 dye due to repeated scanning is minimal. We tried washing with three different liquids: an antibody stripping buffer, that is normally used to disrupt all protein protein interactions in an western blot; water and the binding buffer, that was normally used in our Plasmid Repressor Interaction Assay.

We pursued our second approach which was based on repressors fused to a cellulose binding domain (BBa_K1321340). All constructs were cloned successfully. When pipetted onto various types of paper, its presence could be confirmed by staining and destaining of the paper with Coomassie brilliant blue and destaining solution used for SDS-PAGES (45 % EtOH, 10 % acetic acid in ddH₂O). The binding was unspecific. Any protein could be pipetted onto paper and be detected with Coomassie brilliant blue. Besides, most CBDs bind to microcristalline cellulose or cotton. We were not able to find a hint about the binding of CBDs to common paper. That is why we focused on the approach with immobilized DNA.

Since the repressorproteins we wanted to detect were tagged with sfGFP, they were detectable via fluorescence. DNA was labeled with Cy3 containing primers, therefore it was also detectable on paper. Both components were visualized with the Ettan Dige. The exposure time was optimized as was the paper that was used.

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