Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells

Eiko Sasaki, Yoshifumi Okamoto, Kaya Yoshida, Hirohiko Okamura, Katsuhide Shimizu, Fumio Nasu, Hiroyuki Morimoto, Tatsuji Haneji

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/ streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.

Original languageEnglish
Pages (from-to)327-333
Number of pages7
JournalHistochemistry and Cell Biology
Volume120
Issue number4
DOIs
Publication statusPublished - Oct 1 2003
Externally publishedYes

Keywords

  • Avidin-FITC
  • Avidin-interacting proteins
  • Avidin-peroxidase
  • Cultured cells
  • Mitochondria

ASJC Scopus subject areas

  • Histology
  • Molecular Biology
  • Medical Laboratory Technology
  • Cell Biology

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