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 efficiency was developed for DNA and RNA. The present work assessed the applicability of this method to proteins in a model based on human apolipoprotein(a)[apo(a)] isoforms which consist of a single, >200-kDa polypeptide chain varying in size with a repeat sequence. After reduction treatment and sodium dodecyl sulfate-agarose gel electrophoresis, serum proteins were transferred from the gel by upward or downward (Turboblotter) capillary action onto nitrocellulose membranes in Tris-buffered saline, pH 7.5, at room temperature. Increased detectability of apo(a) isoforms was achieved by substituting comparatively high molar concentrations of protein A for true second antibody. With downward capillary transfer and short 37 degrees C incubations, the apo(a) phenotyping could be completed in about 26 h and required less than 8 h effective processing time. The downward transfer was about twice as fast (complete within 1 h) as the upward version and with this speed it offers a good alternative to electroblotting as well.
Nagy
B
B
Clinical Pathology Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
Costello
R
R
Csako
G
G
D001053:Apolipoproteins / Q000097:blood*; D053241:Apoprotein(a); D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D017270:Lipoprotein(a)
D001053:Apolipoproteins; D017270:Lipoprotein(a); D053241:Apoprotein(a)