Detecting Protein-DNA Interactions
The HaloLink Protein Array System can also be used for protein-nucleic acid interaction studies. Traditionally these interactions are detected using electophoretic mobility shift and fluorescence anisotropy assays. These methods need purified proteins, are labor intensive, and have low throughput. HaloLink, in contrast, is rapid, requires no purified protein, and provides a medium-throughput format.
To show that HaloLink can be used to study protein-nucleic acid interactions we selected: interaction between DNA binding protein p50 and target DNA sequence and inhibition of DNA binding to p50 by IκBα (an ankyrin repeat protein that can weakly bind to the DNA binding domain). One wheat-germ extract cell-free reaction was used for the expression of the HaloTag-p50 fusion protein, while another reaction was used to co-express HaloTag-p50 and IκBα with T7 epitope tag.
Capture of HT-p50 (Figure 3; slide 1) and HT-p50+ IκBα (Figure 3; slide 1 and 2) complex on a HaloLink slide was confirmed by probing with anti HaloTag antibody and anti T7 epitope antibody respectively followed by AlexaFluor 647 labeled secondary antibody. A dilution series of HaloTag-GST at known concentrations was added to the slide and also probed with anti HaloTag antibody (Figure 3, Slide 1).
A HaloTag-GST titration curve provides an estimate of expression level of HaloTag fusion and is used to normalize for day-to-day variations.
On a separate slide, a p50 consensus DNA sequence labeled with AlexaFluor 647 was used to probe p50 and p50-IκBα dimer. The intensity of DNA-AlexaFluor 647 was threefold lower on p50-IκBα dimer compared to p50 protein alone (Figure 3, Slide 3) validating the result that IkBa binding to p50 DNA binding domain reduces the binding efficiency of consensus DNA sequence. This experiment demonstrates the versatility of HaloLink to not only perform protein-DNA interaction experiments but also extract qualitative information about the binding affinities.