When I published a synopsis from Erandi on discovering AP2 binding sites in P. falciparum, I knew at some point I have to write about PBMs. De Silva et al. (2008) have used this ingenious method and apparently it works. The basic idea is that in many cases, we have identified a putative transcription factor and we are interested in finding its binding site (and subsequently identifying its potential targets). This method starts by the purification of an epitope-tagged version of our protein which is then hybridized to a microarray slide harboring all possible k-mers of a given length (e.g. 10-mers). Upon washing away the non-specific interactions, a fluorophore-conjugated antibody (against the epitope-tag) is used to find the spots containing protein-DNA interactions. The final step involves overlapping the bound oligos and finding the consensus binding sequence.
As you see, the theory is simple; however, making the PBM is not trivial given the space of all possible sequences. Below, I have attached a figure from the original paper demonstrating this method. First, they use the notion of de Bruijn sequences to minimize the number of spots needed to represent all possible k-mers. Upon synthesizing these oligos on a slide, they convert them to dsDNA (Cy3 labeled dUTP) using a Cy5 labeled universal primer. The labels are used to ensure that the reactions are in fact completed. An Alexa488-conjugated GST antibody is then used to identify the proteins bound to these features.
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