Various groups employed different binding assays in studying Wnt-Arrow/Lrp5/Lrp6 interactions, which appear to be weaker than Wnt-Fz interactions (M.S. and X.H., unpublished), and the binding affinities have not been reported.

One assay involves the production of conditioned medium (CM) containing either the secreted Arrow/Lrp5/Lrp6 extracellular domain or Wnt/Wg, then incubation of the two CM together and subsequent co-IPs. Note that CM contains bovine serum proteins and other secreted proteins made by the cultured cells,and thus is not a pure source of proteins of interest.

Another assay involves co-transfection of Wnt plus Lrp5/Lrp6, either the full length or just the extracellular domain, followed by co-IPs using total cell extracts. One cautionary note here is that Wnt and Lrp5/Lrp6, because in part of their high cysteine content, may exhibit some misfolded proteins in the secretory pathway when overexpressed (see Box 4). Thus total cell lysates may contain Lrp5/Lrp6 and/or Wnt aggregates that pose problems for co-IPs. This is less a problem when using CM, as it is generally believed that proteins secreted into the CM (or presented on the plasma membrane) have been properly matured through the secretory route, i.e. have been correctly folded and processed.

The third assay tests the binding of the Arrow extracellular domain(provided by CM) to cells expressing a membrane-tethered form of Wg.