Limitation of enzyme-conjugated streptavidin-biotin system in the detection of eukaryotic proteins by immunoblot analysis.
In a series of Western blot experiments, we discovered that peroxidase-conjugated streptavidin alone without pretreatment of primary and secondary antibodies could react specifically with approximately 145 KD and 75 KD proteins. These bands were eliminated by preincubation with nonconjugated streptavidin. They were found in virtually all tissues of the mouse and rat and various cell lines to a variable degree. It is the thought that these bands may represent some members of biotin-coupled carboxylating enzymes. From these results we suggest that the enzyme-conjugated streptavidin system is not suitable to detect the protein in the eukaryotic cells and tissues by immunoblot analysis, especially those whose size is similar to the biotin-coupled carboxylases.
Kin
S H
SH
Department of Pharmacology, College of Medicine, Seoul National University, Korea.
Suh
Y H
YH
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives*; D015153:Blotting, Western / Q000379:methods*; D001923:Brain Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley
D001426:Bacterial Proteins; D011506:Proteins; C066114:biotin-streptavidin complex; D001710:Biotin