Two photo-leucines were incorporated into the critical receptor-binding region of WT b without interfering with its receptor-binding activity, which allowed highly specific zero distance cross-linking of its direct binding partner(s) but not other neighboring molecules. A biotin moiety of WT b facilitated purification of the complex of WT b and its binding partner(s) by streptavidin beads. An mAb, 2D3, was developed to recognize WT b on an epitope outside the receptor-binding site, serving as a highly specific tool for detection as well as additional affinity purification of the complex. Thus, the binding partner(s) was first cross-linked by WT b , and then purified by using streptavidin and 2D3 beads in tandem. The covalent interaction between the WT b ligand and its partner(s) enabled a purification process under high-stringency conditions, and efficient isolation was achieved irrespective of the nature of the binding partner(s) even if it is a membrane protein(s) with multiple transmembrane domains, like NTCP identified here. NTCP (Slc10a1) is the founding member of the SLC10 family of solute carrier proteins. It is a hepatic Na + bile acid symporter and is responsible for cotransportation of sodium and bile acids across cellular membranes to maintain the enterohepatic circulation of bile acids Stieger, 2011) . NTCP is a multiple transmembrane glycoprotein presumed to span the cellular membrane up to 10 times with small extracellular loops (Mareninova et al., 2005; Hu et al., 2011) . It is mainly expressed in the liver (Stieger, 2011) , consistent with the liver tropism of HBV and HDV. NTCP localizes to the sinusoidal (basolateral) plasma membrane of hepatocytes (Stieger et al., 1994) , a location that fits well with its receptor role for blood-borne HBV and HDV. Whereas HBV first attaches to hepatocytes mainly through heparan sulfate (Schulze et al., 2007; Leistner et al., 2008) , our data demonstrate that the interaction between NTCP and L protein of HBV is highly specific, and NTCP is crucial for productive viral entry of hepatocytes. Consistent with previous reports on primary cultures of rat hepatocytes (Liang et al., 1993; Rippin et al., 2001) , NTCP expression rapidly decreased over time in cultured PTHs after isolation. This may at least partially explain the observations that primary hepatocytes typically remain susceptible to HBV infections in vitro for only a few days after isolation from liver tissues (Gripon et al., 1988; Seeger et al., 2007) .