Transblot identification of biotin-containing proteins in rat liver.
Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.
Haneji
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T
Center for Biomedical Research, Population Council, New York, New York 10021.
Koide
S S
SS
D000818:Animals; D001360:Avidin; D001710:Biotin / Q000032:analysis*; D002262:Carboxy-Lyases / Q000032:analysis*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000201:enzymology*; D008297:Male; D010544:Peroxidases; D051381:Rats; D011919:Rats, Inbred Strains
D001360:Avidin; D001710:Biotin; D003101:Collodion; D010544:Peroxidases; D002262:Carboxy-Lyases