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Volumn 6, Issue 10, 2006, Pages 2177-2183

Biotemplated nanopatterning of planar surfaces with molecular motors

Author keywords

[No Author keywords available]

Indexed keywords

FUNCTIONAL MOTOR PROTEINS; MICROTUBE TRANSPORTERS; NANOPATTERNING; PLANAR SURFACES;

EID: 33750512354     PISSN: 15306984     EISSN: None     Source Type: Journal    
DOI: 10.1021/nl060922l     Document Type: Article
Times cited : (50)

References (34)
  • 11
    • 0033525190 scopus 로고    scopus 로고
    • (a) Wild type kinesin-1 (full length drosophila expressed in bacteria and purified as described by Coy, D. L.: Wagenbach, M.; Howard, J. J. Biol. Chem. 1999, 274, 3667-3671) was mixed with rhodamine-labeled microtubules (labeling ratio: 1 rhodamine/3 unlabeled tubulin units) at a kinesin dimer to tubulin molar ratio of 1:8 in BRB80 buffer containing 10 μM taxol and 1 mM AMPPNP (BRB80TM) to form decorated microtubules. To achieve rapid binding and thus avoid cross-linking of microtubules, we performed the mixing while vortexing the solution. To remove any free, unbound kinesin molecules. 400 μL of the resulting solution (32 nM microtubules, 4 nM kinesin dimers) were centrifuged at 130000g in a Beckmann airfuge for 2.5 min. The pellet was resuspended in 150 μL of BRB80TM. (b) A casein-containing solution (0.5 mg/mL in BRB80) was perfused into the flow cell and allowed to adsorb to the surface for 5 min to reduce the denaturation of kinesin and to prevent the sticking of microtubules. Then the solution containing the decorated microtubules was perfused. After 5-10 min, the flow cell was washed with BRB80TM containing anti-bleaching reagents (20 mM D-Glucose, 0.020 mg/mL glucose oxidase, 0.008 mg/mL catalase and 1% 2-mercaptoethanol). To walk the microtubules off the surface, we perfused an ATP anti-bleaching solution (containing 1 mM Mg-ATP instead of AMPPNP) imo the cell. In the last step, motility solution containing microtubules (∼30 nM tubulin) and ATP antibleaching solution was added to the cell.
    • (1999) J. Biol. Chem. , vol.274 , pp. 3667-3671
    • Coy, D.L.1    Wagenbach, M.2    Howard, J.3
  • 14
    • 33750497991 scopus 로고    scopus 로고
    • note
    • Antibody labeling of the kinesin tracks was performed by perfusing 1.25 μg/mL anti-kinesin (mouse monoclonal SUK4; Cytoskeleton) in PB100BA buffer (100 mM phosphate buffer; pH = 7.5, containing 1 mg/mL BSA and 1 mM ATP) into the flow cell instead of the motility solution. After one hour incubation, we rinsed the cell with PB100BA. FITC-labeled goat anti-mouse IgG (whole molecule; 17 μg/mL in PB100BA; Sigma-Aldrich) was added as secondary antibody. After another 45 minutes of incubation, the solution was exchanged with an ATP anti-bleaching solution.
  • 24
    • 33750515165 scopus 로고    scopus 로고
    • note
    • For the cargo transport experiment, we perfused rhodamine-biotin-labeled transport microtubules (labeling ratio: 3 rhodamine/4 biotin/9 unlabeled tubulin units; ∼30 nM tubulin) in AMPPNP anti-bleaching solution into the cell instead of the motility solution. After they had bound to the generated kinesin tracks, streptavidin-coated quantum dots (525 nm emission, 20 000x diluted; Invitrogen) were loaded onto the transport microtubules. Finally, an ATP anti-bleaching solution was added to start the movement of the cargo-loaded transport microtubules.
  • 27
    • 0028899953 scopus 로고
    • Beta-tubulin (TUB 2.1, 4 μg/mL; Sigma) or tetramethylrhodamine (4 μHg/mL; Molecular Probes) antibodies were adsorbed for 5 min onto a hydrophobic glass surface that was previously treated with dichlorodimethylsilane. The surface was then blocked against unspecific protein binding by Pluronic F127 (1% dissolved in BRB80). Rhodamine-biotin-labeled microtubules (labeling ratio: 3 rhodamine/4 biotin/9 unlabeled tubulin units) were polymerized, and unpolymerized tubulin was removed by 5 min centrifugation at 180000g in a Beckmann airfuge. The pellet was resuspended in BRB80 containing 10 μM taxol to a final tubulin concentration of 80 nM.; this solution was added to the flow cell in order to bind these template microtubules to the antibodies on the substrate surface. In the next steps, first fluorescein labeled streptavidin (10 μg/mL in BRB80, Pierce) was perfused into the flow cell. Secondly, biotin-ylated kinesin K401-BIO (Berliner, E.; Young, E. C.: Anderson, K.; Mahtani, H. K.; Gelles, J. Nature 1995, 373, 718-721.) (2.5 μg/mL in BRB80, containing 10 μM taxol, 0.5 mg/mL casein and 20 mM ATP) was added. The flow cell was rinsed with ATP anti-bleaching solution, and microtubule containing motility solution was perfused. In experiments with tubulin antibodies, free tubulin was added to the motility solution with a final concentration of 20 μg/mL. In experiments where tetramethylrhodamine antibodies were used to immobilize template microtubules, the transport microtubules were labeled with Alexa-488.
    • (1995) Nature , vol.373 , pp. 718-721
    • Berliner, E.1    Young, E.C.2    Anderson, K.3    Mahtani, H.K.4    Gelles, J.5
  • 28
    • 33750503723 scopus 로고    scopus 로고
    • note
    • 2) containing 10 μM taxol, 1 mM ATP, and 100 mM NaCl. This solution was centrifuged for 5 min at 180 000g in a Beckmann airfuge to remove clustered molecules and then added to the fixed microtubules in the flow cell. Finally, after washing twice with BRB20 containing 10 μM taxol and 1 mM ATP, motility solution containing microtubules (∼30 nM tubulin) and ATP anti-bleaching solution (in BRB20) was flown in.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.