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SDS-PAGE of kinesin-1 monomers tethered at loop 2 . Untethered monomeric kinesin-1 constructs appear as bands around 38 kDa. Calculated molecular weights for tethered constructs were as follows: 39 kDa (PEG_0.8k), 41 kDa (PEG_2k), 44 kDa (PEG_5k), 45 kDa (20-nt ssDNA), 52 kDa (40-nt ssDNA), 57 kDa (60-nt ssDNA), 45 kDa (20-bp dsDNA), 51 kDa (40-bp dsDNA), and 57 kDa (60-bp dsDNA). Labelling efficiencies were 61–100 % for PEG, 7–45 % for ssDNA, and 22–49 % for dsDNA.

Journal: MethodsX

Article Title: In vitro motility-based tether-scanning of the kinesin motor domain

doi: 10.1016/j.mex.2025.103719

Figure Lengend Snippet: SDS-PAGE of kinesin-1 monomers tethered at loop 2 . Untethered monomeric kinesin-1 constructs appear as bands around 38 kDa. Calculated molecular weights for tethered constructs were as follows: 39 kDa (PEG_0.8k), 41 kDa (PEG_2k), 44 kDa (PEG_5k), 45 kDa (20-nt ssDNA), 52 kDa (40-nt ssDNA), 57 kDa (60-nt ssDNA), 45 kDa (20-bp dsDNA), 51 kDa (40-bp dsDNA), and 57 kDa (60-bp dsDNA). Labelling efficiencies were 61–100 % for PEG, 7–45 % for ssDNA, and 22–49 % for dsDNA.

Article Snippet: The specific DNA sequences used were as follows: 20-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCT-biotin-3′ 40-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT-biotin-3′ 60-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT-biotin-3′ 20-bp dsDNA: 5′-amine-TACTAACTAATCGTCCAACG-3′ 5′-biotin-CGTTGGACGATTAGTTAGTA-3′ 40-bp dsDNA: 5′-amine-CGTTGGACACGACAGGTCCTTAGACTGAAATTAGTTAGTA-3′ 5′-biotin-TACTAACTAATTTCAGTCTAAGGACCTGTCGTGTCCAACG-3′ 60-bp dsDNA: 5′-amine-ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGAGCCATCTGCATGAATGAATCGGCCAACG-3′ 5′-biotin-CGTTGGCCGATTCATTCATGCAGATGGCTCGACAGGTTTCCCGACTGGAAAGCGGGCAGT-3′ As PEG tethers, Biotin-PEG11-amine (0.8 kDa, Tokyo Chemical Industry, Tokyo, Japan), Biotin-PEG2k-Maleimide (2 kDa, Biopharma PEG Scientific), and Biotin-PEG5k-Maleimide (5 kDa, Laysan Bio) were purchased.

Techniques: SDS Page, Construct

In vitro microtubule gliding assay . (a) Scheme of the in vitro microtubule gliding assay. Single-headed kinesins are tethered to the streptavidin-coated glass surface via tethers using biotin-avidin conjugation. (b) Image of fluorescent microtubules bound to surface-tethered kinesin motor domains via 40-bp DNA linkers. (c) Kymographs of gliding-microtubules driven by kinesin motor domains tethered at loop-2 via PEG_5k, 20-nt ssDNA, and 20-bp dsDNA linkers.

Journal: MethodsX

Article Title: In vitro motility-based tether-scanning of the kinesin motor domain

doi: 10.1016/j.mex.2025.103719

Figure Lengend Snippet: In vitro microtubule gliding assay . (a) Scheme of the in vitro microtubule gliding assay. Single-headed kinesins are tethered to the streptavidin-coated glass surface via tethers using biotin-avidin conjugation. (b) Image of fluorescent microtubules bound to surface-tethered kinesin motor domains via 40-bp DNA linkers. (c) Kymographs of gliding-microtubules driven by kinesin motor domains tethered at loop-2 via PEG_5k, 20-nt ssDNA, and 20-bp dsDNA linkers.

Article Snippet: The specific DNA sequences used were as follows: 20-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCT-biotin-3′ 40-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT-biotin-3′ 60-nt ssDNA: 5′-amine-CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT-biotin-3′ 20-bp dsDNA: 5′-amine-TACTAACTAATCGTCCAACG-3′ 5′-biotin-CGTTGGACGATTAGTTAGTA-3′ 40-bp dsDNA: 5′-amine-CGTTGGACACGACAGGTCCTTAGACTGAAATTAGTTAGTA-3′ 5′-biotin-TACTAACTAATTTCAGTCTAAGGACCTGTCGTGTCCAACG-3′ 60-bp dsDNA: 5′-amine-ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGAGCCATCTGCATGAATGAATCGGCCAACG-3′ 5′-biotin-CGTTGGCCGATTCATTCATGCAGATGGCTCGACAGGTTTCCCGACTGGAAAGCGGGCAGT-3′ As PEG tethers, Biotin-PEG11-amine (0.8 kDa, Tokyo Chemical Industry, Tokyo, Japan), Biotin-PEG2k-Maleimide (2 kDa, Biopharma PEG Scientific), and Biotin-PEG5k-Maleimide (5 kDa, Laysan Bio) were purchased.

Techniques: In Vitro, Gliding Assay, Avidin-Biotin Assay, Conjugation Assay