ID 240
Title Transcription
アビジン ケツゴウ タンパク ノ サイボウナイ キョクザイ ニツイテ : ウエスタン ブロッティング ヘンポウ ト サイボウ カガクテキ ホウホウ オ モチイテ
Title Alternative
Localization of Avidin-interacting Proteins in Cultured Cells : Transblot and Cytochemical Detection
Author
Okamoto, Yoshifumi Department of Histology and Oral Histology, Graduate School of Dentistry, The University of Tokushima
Keywords
アビジン
ビオチン
カルボキシラーゼ
ミトコンドリア
ABC法
細胞分画
Content Type
Departmental Bulletin Paper
Description
Cell lysates prepared from 3T3-L1 cells were separated by SDS-PAGE, transferred to PVDF membranes and incubated with peroxidase-conjugated avidin. Peroxidase-conjugated avidin interacted with bands of proteins with estimated molecular weights of 120, 74, and 72 kDa. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these 3 proteins in MC3T3-E1, YROS-1, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Cell lysates prepared from 3T3-L1 cells were also analyzed by avidin-biotin complex Western blotting method using the 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 these 3 proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. However, these proteins were not detected in the fractions containing nucleus or peroxisome. FITC-avidin was also localized in mitochondria in the cultured cells. The localization of avidin-interacting proteins in mitochondria was confirmed by using rhodamine-avidin or by double staining with FITC-avidin and Mito-tracker. When attempting to use avidin-biotin complex immunoblotting and immunohistochemical procedures to detect the specific proteins in cultured cells, the avidin-interacting proteins demonstrated in the present study will be detected and will give non-specific staining if the corresponding concentrations of peroxidaseconjugated avidin are used. The present study also indicates that careful observation should be taken when using the avidin-biotin complex system in biochemical and histochemical examination.
Journal Title
四国歯学会雑誌
ISSN
09146091
NCID
AN10050046
Volume
18
Issue
1
Start Page
27
End Page
37
Sort Key
27
Published Date
2005-06
Remark
公開日:2010年1月24日で登録したコンテンツは、国立情報学研究所において電子化したものです。
FullText File
language
jpn
Report Type
学位論文