DOWNWARD BLOTTING OF PROTEINS IN A MODEL-BASED ON APOLIPOPROTEIN(A) PHENOTYPING

Standard immunoblotting (''Western blot'') involves electrotransfer of proteins from a separation gel (usually acrylamide) onto a membrane. Recently, a downward capillary method with increased hybridization eff iciency was developed for DNA and RNA. The present work assessed the a pplicability of this method to proteins in a model based on human apol ipoprotein(a) [apo(a)] isoforms which consist of a single, >200-kDa po lypeptide chain varying in size with a repeat sequence. After reductio n treatment and sodium dodecyl sulfate-agarose gel electrophoresis, se rum proteins were transferred from the gel by upward or downward (Turb oblotter) capillary action onto nitrocellulose membranes in Tris-buffe red saline, pH 7.5, at room temperature. Increased detectability of ap o(a) isoforms was achieved by substituting comparatively high molar co ncentrations of protein A for true second antibody. With downward capi llary transfer and short 37 degrees C incubations, the apo(a) phenotyp ing could be completed in about 26 h and required less than 8 h effect ive processing time. The downward transfer was about twice as fast (co mplete within 1 h) as the upward version and with this speed it offers a good alternative to electroblotting as well. (C) 1995 Academic Pres s, Inc.