Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells.
An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.
Hanaki
Ken-ichi
K
Japan Society for the Promotion of Science, University of Tokyo, Tokyo, Japan. khanaki@m.u-tokyo.ac.jp
Ohka
Seii
S
Yamamoto
Kenji
K
Nomoto
Akio
A
Yoshikura
Hiroshi
H
D000818:Animals; D002522:Chlorocebus aethiops; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D049690:History, Ancient; D008856:Microscopy, Fluorescence / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011067:Poly dA-dT / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling; D014709:Vero Cells
D009838:Oligodeoxyribonucleotides; D011506:Proteins; C016023:oligo(dA-dT); D011067:Poly dA-dT