User:Steven J. Koch/Notebook/Kochlab/2009/06/05/New antifade components

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See Linh's notes for results. Bottom line: Brigette's new batch of antifade worked very well, while the antifade from earlier this week was not working, and according to Linh's and Igor's work, was possibly even accelerating the breakup of the MTs.

My notes

Stock concentrations:

   * 800 micrograms / ml of catalase (100x)
   * 2 mg / ml of glucose oxidase (100x) 

9.9 mg is the weight of the catalase, so adding 12.4 ml of cold BRB80. This was vortexed. Was put into aliquots of 1 to 1.5 ml and stored at -20C.

(Not sure exactly what Brigette did for the GOD measurements). Approx. 10 milliliters, though. Was vortexed. Was 0.2 micron filtered. Brigette aliquoted.

These new components were used to make new antifiade cocktail. Not sure whether Brigette stored antifade cocktail aliquots. Given our recent results, it will be interesting to know whether the cocktail aliquots work well. (I remember them working well for me at Sandia.)

Filtered the GOD, but the CAT would not go through the filter

MTs

Linh and Igor polymerized a FITC aliquot, and stabilized with BRB80-T. They made new BRB80-T today from PEM + taxol (same taxol aliquot that we resuspended last week). Note: I remember from Sandia that someone told me the Taxol would fall out of BRB80-T and that you should vortex the BRB80T before use Not sure if this is true. It may also be true that you are better off making BRB80-T frequently, not sure.

Andy Maloney 23:58, 5 June 2009 (EDT): You are right to say that taxol will "fall" out of solution if left alone. This is because taxol is insoluble in aqueous buffers. For this reason, we use the delicious and garlic-y DMSO. DMSO is a wonder chemical that is soluble in both polar and non-polar solvents. If taxol falls out of solution, then this makes me think that DMSO has a higher affinity for polar solvents than it has for non-polar solutes. At any rate, it is a good idea to vortex everything you use that has been sitting for some time. Just from past experiences.