User:Brigette D. Black/Notebook/Brigettes Notebook/2009/09/14/What's wrong with all these assays?!?

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Andy and I have both been suffering from a complete lack of activity. The common factor we seemed to find was the PEM-T we had been using, and potentially the "goodness" of the entire batch of PEM that was made on 8/22.

So today I did a very quick and dirty kinetics assay using PEM-T that I made from old stock (in the -20 freezer, has a PT on the top of it instead of just a T). This is a different stock than the taxol we have been using recently.

I added 2.5 uL of the 2mM taxol solution to 497.5 uL of PEM buffer made in June, and the most recent batch in July.

I polymerized 2 aliquots of unlabeled tubulin, and stablized one with the June PEM-T, and the other with the August PEM-T (used 195 uL PEM-T for both).

I then added 0.4 uL of 100mM MgATP and 0.2 uL of BME (1mM ATP + 0.5 mM BME per 40 uL reaction) to each of 10 aliquots. I then pipetted in 10 uL of the "June MTs" into aliquots 1-5, "August MTs" into aliquots 6-10. I then added 30 uL of kinesin at 0.83 ug/ml (diluted with "June" PEM and "August PEM).

  • Cuvette 1: June, 0 min
  • Cuvette 2: June, 10 min
  • Cuvette 3: June, 20 min
  • Cuvette 4: June, 30 min
  • Cuvette 5: June, 45 min
  • Cuvette 6: August, 0 min
  • Cuvette 7: August, 10 min
  • Cuvette 8: August, 20 min
  • Cuvette 9: August, 30 min
  • Cuvette 10: August, 45 min

After letting them react for the appropriate time, I quenched the solutions, and the let the malachite green dye react for 16 minutes.

I do have results, but no plots made or analysis yet. Coming soon!

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