User:Brigette D. Black/Notebook/Brigettes Notebook/2009/09/21/New Kinesin
Processes: Go off of motility assay model
So, dilute the borrowed kinesin 1:15 (20 uL kinesin to 300 uL PEM) MTs:
5uL (@5 mg/ml) +195 uL PEM-T (.125 mg/ml) = (1.369*10^15 kDa/mL) = (2.4885 *10^13 molecules/mL) =(4.132348 *10^-11 mols/mL) = (4.132348*10^-8 mols/L) = 41.32348 nM
We want ~32 nM, so the concentration above seems pretty good.
What I did:
20 uL kinesin to 300 uL PEM (1:16)
MTs: (5uL +195+288+2 +10) = .05 mg/ml (16.53 nM)
ATP: (10 uL at 100mM = 2mM in MT solution) in cuvette: 1mM BME: (2 uL into MTs)
So, about half as many MTs as specified, and just a little less kinesin.
Mixed 300 uL of the MT mixture with 300 uL of the diluted kinesin. Let react in a 1.5 mL aliquot at room temp covered with foil, and gently agitated aver 15 minutes or so (I had a crazy idea that maybe the since the MTs are heavier than the kinesin they might fall to the bottom and make reacting impossible).
Quenched 40 uL of the reaction with 200uL of perchloric acid, then added 200uL Malachite green dye and let develop for 16 minutes.
Reactions: MT (0) MT+K(0 min) MT+K(10 min) MT+K(20 min) MT+K(30 min) MT+K(40 min) MT+K(50 min) MT+K(60 min) MT (60 min) MT+K(75 min) MT+K(90 min) MT+K(105 min) MT+K(120 min) MT(120 min)
- -( NOT AWESOME :-( In fact, not even decent. Totally sucks. I'll post a graph in a bit.