Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots

Marco Tjioe, Hyeon Ryoo, Yuji Ishitsuka, Pinghua Ge, Carol Bookwalter, Walter Huynh, Richard J. McKenney, Kathleen M. Trybus, Paul R Selvin

Research output: Contribution to journalArticle

Abstract

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 μg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.

Original languageEnglish (US)
Pages (from-to)2278-2286
Number of pages9
JournalBioconjugate Chemistry
Volume29
Issue number7
DOIs
StatePublished - Jul 18 2018

Fingerprint

Quantum Dots
Cytoskeleton
Microtubules
Semiconductor quantum dots
Purification
Kinesin
Dyneins
Centrifugation
Labels
Proteins
Fluorophores
Assays
Sediments
Molecules

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots. / Tjioe, Marco; Ryoo, Hyeon; Ishitsuka, Yuji; Ge, Pinghua; Bookwalter, Carol; Huynh, Walter; McKenney, Richard J.; Trybus, Kathleen M.; Selvin, Paul R.

In: Bioconjugate Chemistry, Vol. 29, No. 7, 18.07.2018, p. 2278-2286.

Research output: Contribution to journalArticle

Tjioe, M, Ryoo, H, Ishitsuka, Y, Ge, P, Bookwalter, C, Huynh, W, McKenney, RJ, Trybus, KM & Selvin, PR 2018, 'Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots', Bioconjugate Chemistry, vol. 29, no. 7, pp. 2278-2286. https://doi.org/10.1021/acs.bioconjchem.8b00264
Tjioe, Marco ; Ryoo, Hyeon ; Ishitsuka, Yuji ; Ge, Pinghua ; Bookwalter, Carol ; Huynh, Walter ; McKenney, Richard J. ; Trybus, Kathleen M. ; Selvin, Paul R. / Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots. In: Bioconjugate Chemistry. 2018 ; Vol. 29, No. 7. pp. 2278-2286.
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