3.3. Pull-Down Assays with Albumin-Conjugated Resins

Human and mouse serum albumin (HSA and MSA, respectively; Sigma Aldrich, Atlanta, GA, USA) and BSA were dissolved in PBS (final concentration, 2.5% (w/v)) and mixed with glutaraldehyde (Sigma Aldrich) at a final concentration of 0.2% (v/v) to allow polymerization. After incubation at room temperature for 4 h, mixtures were dialyzed against PBS overnight at 4 °C. Subsequently, 100 µg of HSA, MSA, BSA, as well as polymerized HSA, MSA, and BSA (pHSA, pMSA, and pBSA, respectively), were individually coupled to 200 µL of N-hydroxysuccimide (NHS)-activated Sepharose 4 Fast Flow (GE Healthcare, Buckinghamshire, UK; 50% slurry). PAR-deleted BNCs (ΔBNCs, BNCs lacking a large part of the pre-S region from Met-1 of the pre-S1 region to Arg-18 of the pre-S2 region [40]) were prepared by incubating BNCs with 0.2% (w/w) trypsin (Sigma Aldrich) at 37 °C for 1 h. Each albumin-conjugated resin (20 µL) was incubated with 40 µg of either BNCs or ΔBNCs in 100 µL of PBS at 37 °C for 1 h, washed with PBS 4 times, subjected to 0.1% (w/v) sodium dodecyl sulfate-12% (w/v) polyacrylamide gel electrophoresis (12% SDS-PAGE), followed by silver staining. The amounts of BNCs and ΔBNCs bound to albumin-conjugated resins were determined by densitometry using a luminescent image analyzer (LAS-4000mini; Fujifilm, Tokyo, Japan).