The implementation of small-angle X-ray scattering (SAXS) in combination with size exclusion chromatography (SEC) has provided a useful tool for increasing the purity of samples during SAXS data collection. With the use of SEC-SAXS, samples become diluted and so methods to enhance the scattering signal of the samples at lower concentrations have been applied to beamline B21 using a modified sample cell, a variety of window materials and longer exposure times. SEC-SAXS at B21 provided a method of collecting data for biological macromolecules where aggregation and low concentration issues are a major problem. This method of data collection is shown with a variety of proteins, including matrix proteins, which are less amenable to SAXS data collection. Bone morphogenetic proteins (BMP) are essential signalling molecules involved in developmental and pathological processes and are regulated in the matrix by secreted glycoproteins. One such regulator is BMP-binding endothelial cell precursor-derived regulator (BMPER) which can both inhibit and enhance BMP signalling in a context and concentration-dependant manner. Twisted gastrulation (Tsg) also has pro- and anti-BMP properties but it is unclear whether Tsg and BMPER directly interact and whether they act synergistically on BMP signalling. Here, we show that BMPER binds to Tsg with high-affinity through the N-terminal region of BMPER. Cell-based inhibition assays show that the N-terminal region of BMPER is a better inhibitor of BMP-4 signalling than full-length BMPER. Furthermore, BMPER and Tsg cooperatively inhibit BMP-4 signalling suggesting they act in concert. Full-length BMPER, but not the N-terminal region, binds heparan sulphate (HS) proteoglycans at the cell surface. A disease-causing BMPER point mutation, P370L, which is found in the acid-catalysed internal cleavage site, was introduced to BMPER. The mutation caused a change to the location of the cleavage site, decreased HS-binding capability and increased BMP inhibition. These data suggest that localisation of BMPER at the cell surface reduces BMPER activity and consequently BMP inhibitory potential, which may be ameliorated by the mutation. To interrogate how these regions within BMPER are arranged, small-angle X-ray scattering and electron microscopy were used to show that BMPER is elongated which spaces the N-terminal BMP-binding and C-terminal cell-interactive regions.