RATE electrophoresis of NMR and human plasma and analysis of the receptor-binding properties of their A2M.

<p>RATE electrophoresis was done using 50 μg native NMR and human plasma and 50 μg of methylamine and trypsin treated plasma, respectively. Isolated human A2M served as control. Methylamine as well as trypsin shift human and NMR-A2M from the slow- to the fast-moving form. To visualize the respective protein bands the gel was stained with Commassie (A). Purified LRP1 (100–500 ng) was spotted to a nitrocellulose membrane and incubated with either 10 μg/mL human or NMR plasma. BSA served as negative control. A2M binding to LRP1 was detected by a polyclonal rabbit anti-human A2M antibody (5 μg/mL) (B). To block the binding of A2M to LRP1 the spotted LRP1 was pre-incubated with 1.5 μM RAP for 30 min prior to the addition of human or NMR plasma (C). (☐ - native/slow form of A2M, ◆ –transformed/fast form of A2M).