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