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We bought commercial Kinesin Heavy Chain Motor Domain (H. sapiens recombinant) from Cytoskeleton . I re-suspended the protein in CRB (CMW Buffer) with a final concentration of 5μg/μL, as suggested by the product data sheet. (Adding 5 μL of CBR to a 25 μg of KHC vial)
Originally, the Kinesin was diluted to 27.5 μg/mL for each assay. So, to achieve this I had to dilute 0.55 μL of KHC in CBR with 99.45 μL of CBR.
I run the assay in the very same way I used to run it with the “old” Kinesin. Here are the results:
This has an average of 71.2 nm/s.
This result is nowhere near the velocities we observed with the “old” Kinesin (~ 900-1000 nm/s). There are many possible factors that could have caused this. The most important is the fact that both have been extracted from different organisms, and some mutations would be expected. Nevertheless, I would not expect such a big difference.
Another additional factor would be the fact that we used one buffer (CRB) to reconstitute the protein and another (PEM) to complete the assay. However, to be quite honest; I am not sure what buffer was used to reconstitute the “old” Kinesin.