Blot overlay assay of r Pf M18AAP and erythrocyte membrane proteins. Blot overlay assays were performed four times under hypotonic (A) and isotonic (B) conditions and illustrate the interaction between rPf M18AAP and erythrocyte membrane skeleton proteins. Blot overlays are in the left panels of each figure and the Laemmli SDS-polyacrylamide gels are on the right. A. In the absence of salt, and using a short exposure time of 5 minutes for the chemiluminescence reaction, rPf M18AAP binds to beta spectrin, protein 4.1, protein 4.2, actin and G3PD (lane 3 on the immunoblot). Lane 1 – rPf M18AAP (positive control); lane 2 – bovine serum albumin (negative control); lane 3 – erythrocyte membrane proteins. B. In the presence of a physiological salt concentration, lane 2 on the blot overlay on the left depicts a short exposure (5 minutes) and shows that rPf M18AAP binds strongly to alpha spectrin, beta spectrin and G3PD. The reaction with beta spectrin is more intense than with alpha spectrin. Lane 2 on the middle blot overlay depicts a longer exposure (20 minutes) and shows that rPf M18AAP also binds to protein 4.1, protein 4.2 and actin. Lane 1 – rPf M18AAP (positive control); lane 2 – erythrocyte membrane proteins.