Fig. 3.

Effect of Tween 20 on C176 binding to HDL. (a) Affinity chromatography analysis. Affinity chromatography columns immobilized with rScl constructs and exposed to HDL were washed with and without Tween 20-containing buffers. Eluted samples were analyzed by 15% SDS-PAGE. rScl1s are marked with solid triangle and ApoAI is marked with open triangle. (b) Detection of HDL and rScl1 constructs by dot immunoblotting. Protein samples shown in A were spotted onto a nitrocellulose membrane. Presence of ApoAI was tested with anti-ApoAI antibody (bottom row) and rScl1s were detected with a Strep-Tactin-HRP conjugate (upper row). Immunoreactivity was visualized using chemiluminescence substrate. (c) Tween 20 inhibits the interaction of C176V with HDL by ELISA. Different concentrations of C176V were immobilized onto microplate wells and 0.5 μg of HDL was added to the wells. Wells were washed with buffer containing (TBST) or lacking Tween 20 (TBS). Bound HDL was detected with the anti-ApoAI antibody and the secondary antibody conjugated to HRP. The color was developed with an HRP substrate and absorbance was recorded at 450nm. Mean absorbance values±SD from triplicate wells were obtained after subtracting the OD values of the control wells without C176V.