Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays

Matthew P. Nicholas, Lu Rao, Arne Gennerich

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety ofmicroscopy experiments in which MTs are used as substrates for MT-associated proteins, such as themolecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in whichaminosilanized glass is formylated using the cross-linker glutaraldehyde, fl uorescence-labeled MTs arecovalently attached, and the surface is passivated with highly pure beta-casein. The technique presentedhere yields rigid MT immobilization while simultaneously blocking the remaining glass surface againstnonspecifi c binding by polystyrene optical trapping microspheres. This surface chemistry is straightforwardand relatively cheap and uses a minimum of specialized equipment or hazardous reagents. Thesemethods provide a foundation for a variety of optical tweezers experiments with MT-associated molecularmotors and may also be useful in other assays requiring surface-immobilized proteins.

Original languageEnglish (US)
Pages (from-to)137-169
Number of pages33
JournalMethods in molecular biology (Clifton, N.J.)
Volume1136
DOIs
StatePublished - 2014
Externally publishedYes

Fingerprint

Microtubules
Immobilization
Glass
Optical Tweezers
Cytoplasmic Dyneins
Immobilized Proteins
Kinesin
Microtubule-Associated Proteins
Polystyrenes
Glutaral
Caseins
Microspheres
Membrane Proteins
Equipment and Supplies

Keywords

  • Aminosilane functionalization
  • Chemical cross-linking
  • Fluorescence labeling
  • Glutaraldehyde
  • Microtubule motor proteins
  • Microtubules
  • Plasma cleaning
  • Protein immobilization
  • Silanization
  • Single-molecule assays

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

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abstract = "Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety ofmicroscopy experiments in which MTs are used as substrates for MT-associated proteins, such as themolecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in whichaminosilanized glass is formylated using the cross-linker glutaraldehyde, fl uorescence-labeled MTs arecovalently attached, and the surface is passivated with highly pure beta-casein. The technique presentedhere yields rigid MT immobilization while simultaneously blocking the remaining glass surface againstnonspecifi c binding by polystyrene optical trapping microspheres. This surface chemistry is straightforwardand relatively cheap and uses a minimum of specialized equipment or hazardous reagents. Thesemethods provide a foundation for a variety of optical tweezers experiments with MT-associated molecularmotors and may also be useful in other assays requiring surface-immobilized proteins.",
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AU - Nicholas, Matthew P.

AU - Rao, Lu

AU - Gennerich, Arne

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Y1 - 2014

N2 - Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety ofmicroscopy experiments in which MTs are used as substrates for MT-associated proteins, such as themolecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in whichaminosilanized glass is formylated using the cross-linker glutaraldehyde, fl uorescence-labeled MTs arecovalently attached, and the surface is passivated with highly pure beta-casein. The technique presentedhere yields rigid MT immobilization while simultaneously blocking the remaining glass surface againstnonspecifi c binding by polystyrene optical trapping microspheres. This surface chemistry is straightforwardand relatively cheap and uses a minimum of specialized equipment or hazardous reagents. Thesemethods provide a foundation for a variety of optical tweezers experiments with MT-associated molecularmotors and may also be useful in other assays requiring surface-immobilized proteins.

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