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. 2015 Dec 10;5:18085. doi: 10.1038/srep18085

Figure 1. Basic BH-MT tethering strategy and preparation of assay constituents.

Figure 1

(a) Motorized cargos are functionalized with WT hKIF5A while bead handles are functionalized with non-motile mutant hKIF5A (E237A). Construct sequences for kinesin-1 are otherwise identical, consisting of full length KHC and a HIS6 tag at the C-terminus. (b) BHs are specifically bound to E237A hKIF5A via a biotin-neutravidin-biotin sandwich (B-N-B in the figure) functionalized with a Nickel-activated tris-NTA molecule that binds with high affinity to the HIS6 tag on the E237A hKIF5A construct. Protein structure shows kinesin-1 motor domain. E237A mutation is highlighted in red and is in close proximity with a nucleotide (cyan). (c,d) BHs and MCs are prepared separately (see Methods), then combined together with MTs in assay buffer and finally are introduced into a flow chamber to allow filament network assembly.