2004 Fiscal Year Final Research Report Summary
High-Resolution Atomic Force Microscopy for Complex Structure of Protein Molecules
| Project/Area Number |
14598004
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
ポストゲノムのナノサイエンス
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| Research Institution | Keio University |
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Principal Investigator |
FURUNO Taiji Keio University, School of Medicine, Professor, 医学部, 教授 (00165490)
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| Project Period (FY) |
2002 – 2004
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| Keywords | Atomic force microscope / AFM / Streptavidin / Two-dimensional crystal / Protein chip / Adsorption / Immobilization |
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| Research Abstract |
Immobilization of protein molecules onto a substrate is essentially important for high-resolution imaging by AFM. In the present study we exploited the surface of two-dimensional crystal of streptavidin prepared at the air/water interface for this purpose. Several kinds pf proteins were biotinylated and bound to that crystal surface. A prominent achievement in this study is that high -resolution and -contrast imaging of molecular array in 2D streptavidin crystal became always possible.
The number of biotinylated ferritin and catalase bound to the streptavidin crystal was counted in AFM images. The purpose of this experiment was to confirm the usefulness of digital analysis of protein binding by means of direct AFM imaging. The result was that the binding of non-biotinylated protein onto streptavidin crystal was low and digital counting was effective. On the other hand, immobilization via biotinylation did not always afford high-resolution AFM imaging. Further development of specimen preparation for high-resolution imaging was concluded to be necessary. Furthermore, the precision of AFM digital counting was elucidated to be affected by perfection of the crystal of streptavidin. Then the latter half of this research period was devoted to the study of improvement of crystallinity.
Our AFM was conjugated with an inverted optical microscope by mounting the head portion onto the newly designed automatic stage on the inverted optical microscope. With this system we confirmed that imaging protein arrays prepared on a glass cover slip is possible, which implied that the specimen could be observed simultaneously with the optical microscope.
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