User:Brigette D. Black/Notebook/Brigettes Notebook/2009/09/11/Optimal Kinesin/Microtubule Fail
The trouble with the kinetics assays is that it is very difficult to estimate the ratio of kinesin and microtubules that should be used to give optimum results. We want the number of kinesin low enough so that they can freely walk along a microtubule without bumping into one another, but we also want a number high enough to produce a measurable amount of phosphate. The difficulty here is compounded by our uncertainty in the size and length of the microtubules.
I attempted to run a test to find this optimal ratio by measuring various concentrations of microtubules and kinesin. I started out with 0.25 ug/mL kinesin (0.25 uL at 1 1mg/ml diluted in 1mL PEM). I polymerized a 5uL aliquot of unlabeled tubulin, stabilized with 189 uL BRB80T, then added 4uL of MgATP(at 100mM) and 2uL of BME.
I then added varying amounts of kinesin into cuvettes, then added the microtubules+ATP mixture. I let these react for about 30 minutes and then quenched. I let the malachite green mixture develop for 16 minutes before making a measurement.
For each cuvette I let react, I made another that I quenched immediatly so I could measure the background.
- Cuvette 1: 2 uL MT+ 38 uL kinesin (6.25 uM + 0.238 uM; 26.3:1)
- Cuvette 2: 8 uL MT+ 32 uL kinesin (25 uM + .2 uM; 125:1)
- Cuvette 3: 15 uL MT+ 25 uL kinesin (46.877 uM + 0.156 uM; 300:1)
- Cuvette 4: 20 uL MT+ 20 uL kinesin (62.5 uM + 0.125 uM; 500:1)
- Cuvette 5: 25 uL MT+ 15 uL kinesin (78.125 uM + 0.094 uM; 833:1)
- Cuvette 6: 30 uL MT+ 10 uL kinesin (93.75 uM + 0.0625 uM; 1500:1)
- Cuvette 7: 2 uL MT+ 38 uL kinesin + 30 minutes (6.25 uM + 0.238 uM; 26.3:1)
- Cuvette 8: 8 uL MT+ 32 uL kinesin+ 30 minutes (25 uM + .2 uM; 125:1)
- Cuvette 9: 15 uL MT+ 25 uL kinesin + 30 minutes(46.877 uM + 0.156 uM; 300:1)
- Cuvette 10: 20 uL MT+ 20uL kinesin+ 30 minutes (62.5 uM + 0.125 uM; 500:1)
- Cuvette 11: 25 uL MT+ 15 uL kinesin+ 30 minutes (78.125 uM + 0.094 uM; 833:1)
- Cuvette 12: 30 uL MT+ 10 uL kinesin+ 30 minutes (93.75 uM + 0.0625 uM; 1500:1)
The ratio at the end is a ratio to the number of tubulin to kinesin. In for each microtubule we have approximately 1.3*10^5 tubulin. So a ratio of 500:1 is saying that for each 250 tubulin dimers, there in one kinesin molecules.
When I measured these, I found that the background was EXACTLY the same as the reacted measurements!!! So something went wrong.... It occurs to me now that I added the ATP only to the microtubules, so the concentration of ATP is actually lower in cuvettes with a smaller MT concentration. Also, it turns out I only added enough to make the final concentration .5 mM anyway.
So I think I might chalk this one up to my mistake of measuring. However, even with a low ATP concentration, SOME activity should have been present... right? We'll see! (Steve Koch 13:58, 15 September 2009 (EDT): Yes, even at 500 micromolar ATP, you should have substantial activity.)