A double labeling procedure for lipoproteins: independent visualization of dual ligand-receptor interaction with colloidal gold- and 125I-labeled ligands.

The analysis of multiple ligand binding to a single receptor molecule poses a methodological challenge. The chicken oocyte 95-kDa receptor for the uptake of the two major yolk lipoprotein precursors very low-density lipoprotein (VLDL) and vitellogenin (VTG) is such a multipotent transport receptor. Here we describe methods for rapid independent and simultaneous analysis of VLDL and VTG binding to this receptor, termed VLDL/VTG receptor. First, further development of a one-step labeling protocol for chicken lipoproteins with colloidal gold (Au) to visualize independently the binding of VLDL and VTG to the chicken VLDL/VTG receptor is reported. The advantage of this protocol is that the preparation of the Au-lipoprotein conjugates is rapid, and utilization of Au-lipoprotein complexes in ligand blots does not require their further purification, while signal enhancement by silver staining is still applicable. Second, the simultaneous use of 125I- and Au-labeled ligands in a one-step ligand blotting procedure facilitates the direct demonstration of the competitive interaction of VLDL and VTG with the receptor. Following sequential processing of the nitrocellulose strips, binding of differently labeled ligands can be studied independently. In summary, we describe a procedure for differential labeling of lipoproteins and its application toward the analysis of receptors that bind more than one ligand.