User:Nadiezda Fernandez-Oropeza/Notebook/Notebook/2012/02/07

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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.
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.
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:Steve says: for the repeat I would change a couple things: (1) don't dilute as much.  Maybe 0.55 ul into 40 uL of dilution buffeer. AND (2) I wouldn't dilute with CBR, I'd dilute with your normal PEM-alpha.  Also (3) if you buy more, I would try resuspending the kinesin with D2O and D2O as well for dilution.
===Analysis===
===Analysis===
I run the assay in the very same way I used to run it with the “old” Kinesin. Here are the results:
I run the assay in the very same way I used to run it with the “old” Kinesin. Here are the results:

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Commercial Kinesin

  • So far we have been working with truncated Kinesin-1from Drosophila melanogaster provided by Dr. Haiqing Liu.

We bought commercial Kinesin Heavy Chain Motor Domain (H. sapiens recombinant) from Cytoskeleton [1]. 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.

Steve says: for the repeat I would change a couple things: (1) don't dilute as much. Maybe 0.55 ul into 40 uL of dilution buffeer. AND (2) I wouldn't dilute with CBR, I'd dilute with your normal PEM-alpha. Also (3) if you buy more, I would try resuspending the kinesin with D2O and D2O as well for dilution.

Analysis

I run the assay in the very same way I used to run it with the “old” Kinesin. Here are the results:

Commercial KHC
Commercial KHC

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.



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