User:Brigette D. Black/Notebook/Brigettes Notebook/2009/10/21/Motility Assay Attempt
Today Andy informed me that the motility assays were no longer working, both with the Heavy PEM and the regular PEM. So to try to narrow the problem down, I made 21 new aliquots of antifade (5uL a piece in the -20 C freezer). A test of the new antifade on some MTs Andy had polymerized on Tuesday showed that the MTs were depolymerizing pretty rapidly and fading very quickly.
We then went on to make some new D-glucose. I weighed out .14 mg of D-glucose, then added 1.1 mL of PEM (final volume was about 1.3 mL since the the powder took up quite a bit of volume). I then vortexed the aliquot until all the chunks broke apart, then filtered through a 0.2 um filter. A test of this with the new antifade on plain MTs showed a pretty significant improvement. We saw that the MTs were pretty stable and did not fade as quickly as they had been.
Andy had to leave, but I decided to stick around and try a motility assay with all the new ingredients. Unfortunately, I did not see anything. I did see a lot of really hazy MTs that quickly faded out of view, but no motion. So, I think we have narrowed the problem down to the ATP or the kinesin.
I did notice today that there was some thick milky-looking substance in the ATP I pulled out of the freezer. This substance had fallen to the bottom and was covered by just clear regular looking solution. I mentioned this to Andy who had no idea what it was. Is it possible that this ATP gunk only affects kinesin and not MTs? Regardless, it is something to possibly consider and perhaps pull out a new aliquot of ATP to see if it does the same thing.