User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/19/Commercial Kinesin, Kappa Casein, Assay Attempts

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Making Commercial Kinesin

SJK 01:47, 23 June 2009 (EDT)
01:47, 23 June 2009 (EDT)
Kinesin was from cytoskeleton, the first (and only) that we've ordered. Note: if you want, you can try with one of the aliquots of known value. Also, I ordered kits for testing ATPase activity of kinesin in bulk (ensemble) assays. These will be really good to learn how to do as well. I didn't use kits when I did it at Sandia, so I'm hoping it's easier. It will require us using Osinski's spectrophotometer, which Nathan said he'd show us how to use.

Today I wanted to try the motility assay with the new kinesin. I reconstituted the kinesin with:

  • 1mM ATP
  • 5mM BME
  • 200mM KCl
  • ~ 1M BRB80

This worked out to

  • 1uL ATP (at 100mM)
  • 20uL KCl (at 1M)
  • 0.5uL BME (at >99%)
  • 78.5uL BRB80

Total: 100uL

I added 5uL of this buffer to the kinesin, them pipetted 1 uL of the resuspended kinesin into 5 aliquots, then flash froze these in liquid nitrogen, and then placed in the -80C freezer. Just to note, there are 5 aliquots in the freezer, however the fifth (which is unmarked but with the marked aliquots) contains less than 1uL, getting the very last out of the vial was tricky and resulted in a lot of bubbles in the pipette. Also, there was some (maybe .1-.5 uL) of kinesin left in the vial, so I flash froze that and used it for the assay done later in the day.

We needed to dilute the kinesin to 0.05 - 5 ug/ml (in Koch's notes) with BRB80CA, which is 1000 times less than what is frozen in the aliquots. Now, this is where the question of exactly how much kinesin I have factors in.

Attempt 1

I assumed I had a very small amount of kinesin and added 100uL of BRB80CA. This amount would produce a concentration of about 5 ug/mL, which would be the high end of Koch's recommendation.

We made the assay (as described in June 16 notes). What we saw was many microtubules sticking to the cover glass and no directed motion. This was different from previous assays we had looked at where then microtubules were sticking with only one end, not laying completely flat against the glass.

Attempt 2

I added another 100 uL of BRB80CA, for a total of 200 uL. I was hoping that a more dilute kinesin would produce some better results. This should be between 1 - 5ug/mL, which is still well in the range of Koch's procedure.

The results were the same as the first attempt described above

Attempt 3

We were given permission from Koch to use the kappa casein.

From here we diluted the stock casein with BRB80CS0.5 to 0.5 mM

Steve Koch 00:59, 23 June 2009 (EDT): This part is confusing to me...did you dissolve the kappa-casein in BRB80CS0.5? What does the 0.5 mM refer to? Also, it was a mistake for me to make you think you needed my permission to try the kappa casein. It was more just that Andy had discovered the paper on kappa casein, and I didn't want to step on his toes. Then I thought he'd be happy to have you try it.

Also, I thought part of the problem could be that we did not add enough ATP to the kinesin. I added 1uL of 100 mM MgATP to the kinesin to bring the total concentration of 1.5 mM.

The results were not spectacular, again, Many microtubules were sticking to the cover glass and we did not see any directed motion.

Attempt 4

I added another 100uL of BRB80CA to the kinesin, for a total now of 300uL. I added another 1uL of 100mM ATP (2uL additional total) for a final concentration of about 1.7 mM. I was hoping that diluting the kinesin even more (in the 0.1 - 1 mM range) and adding more ATP would help

Results: pretty much the same as above. Sigh. Hopefully we can try this again on Monday and figure out what went wrong!