Improved chemiluminescent western blotting procedure.
A chemiluminescent Western blotting procedure and its application in assays for human transferrin and human immunodeficiency virus-I antibodies are described. The procedure is based on a chemiluminescent substrate, adamantyl 1,2-dioxetane aryl phosphate and alkaline phosphatase-labeled detection antibodies. Different membranes (polyvinylidene fluoride, nitrocellulose, nylon) and a proprietary membrane treatment agent (Nitro-Block) have been studied. This sensitive blotting procedure utilizing AMPPD, a polyclonal rabbit anti-transferrin:goat anti-rabbit IgG-alkaline phosphatase detection complex, and a PVDF membrane blocked with Nitro-Block permits the detection of 125 pg (1.6 fmol) of human transferrin. A novel 1,2-dioxetane substrate, CSPD, has also been evaluated.
Bronstein
I
I
Tropix, Inc., Bedford, MA 01730.
Voyta
J C
JC
Murphy
O J
OJ
Bresnick
L
L
Kricka
L J
LJ
D000218:Adamantane / Q000031:analogs & derivatives; D015153:Blotting, Western / Q000379:methods*; D003101:Collodion; D015483:HIV Antibodies / Q000032:analysis; D006801:Humans; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D015394:Molecular Structure; D009757:Nylons; D011145:Polyvinyls; D014168:Transferrin / Q000032:analysis* / Q000302:isolation & purification; D005465:Fluorocarbon Polymers
D000218:Adamantane; D003101:Collodion; D015483:HIV Antibodies; D008567:Membranes, Artificial; D009757:Nylons; D011145:Polyvinyls; D014168:Transferrin; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers