Detection of biotinylated proteins in polyacrylamide gels using an avidin-fluorescein conjugate.
Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin-fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin-biotin technology.
Nakamura
Michihiro
M
Department of Anatomy and Cell Biology, School of Medicine, Tokushima University, 3-18-15 Kuramoto, Tokushima 770-8503, Japan.
Tsumoto
Kouhei
K
Ishimura
Kazunori
K
Kumagai
Izumi
I
D001360:Avidin / Q000031:analogs & derivatives* / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D019921:Biotinylation / Q000379:methods; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry; D018929:Cell Culture Techniques; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005452:Fluoresceins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D012097:Repressor Proteins; D012680:Sensitivity and Specificity; D016622:Silver Staining; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005452:Fluoresceins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D011506:Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C049445:fluorescein-avidin; D001360:Avidin; D001710:Biotin; D005982:Glutathione Transferase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli