User:Brigette D. Black/Notebook/Brigettes Notebook/2009/07/07/Measuring Phosphate with Malachite Green: Variable ATP
Today I did the first if what will surely be a series of measurements using kinesin and microtubules. I wanted to keep these measurements as close as possible to the reactions that we have in the gliding motility assay.
- Polymerize 5 uL unlabeled tubulin
- Stabilize microtubules with 95 uL BRB80T (total 100uL)
- Make 10 x Motility solution (w/o casein and ATP) (850 uL BRB80 + 25 uL antifade + 100 uL MTs)
I pulled out of the freezer 1 uL of kinesin at 2.5 mg/mL (diluted and then refrozen about 2 weeks ago). I diluted this down to 5 ug/mL by adding 500 uL of BRB80.
I then made the following mixture in cuvettes. Note, each measurement require 60 uL of reactors to each 300 uL of quencher and activator, so all reactions are 60 uL in volume.
- Cuvette 1: 30 uL MTs + 30 uL BRB80
- Cuvette 2: 30 uL MTs + 30 uL BRB80 + 1mM ATP (.6 uL)
- Cuvette 3: 30 uL MTs + 30 uL BRB80 + 2mM ATP (1.2 uL)
- Cuvette 4: 30 uL MTs + 30 uL BRB80 + 3mM ATP (1.8 uL)
- Cuvette 5: 30 uL MTs + 30 uL Kinesin (at 5ug/mL)
- Cuvette 6: 30 uL MTs + 30 uL Kinesin (at 5ug/mL) + 1mM ATP (.6 uL)
- Cuvette 7: 30 uL MTs + 30 uL Kinesin (at 5ug/mL) + 2mM ATP (1.2 uL)
- Cuvette 8: 30 uL MTs + 30 uL Kinesin (at 5ug/mL) + 3mM ATP (1.8 uL)
The first 5 cuvettes are used as background readings and as a way to approximate the amount of phosphate in the unlabeled MTs, ATP, and kinesin before reaction. I let the reactions occur in the cuvette for about 30 minutes (I didn't time it), then added 300uL perchloric acid (0.6 M) to stop the reactions, the 300 uL of the malachite green activator solution. I let the activator develop for 12 minutes before putting them in the spectrophotometer.
- Cuvette 1: 0.01406
- Cuvette 2: 0.06684
- Cuvette 3: 0.06753
- Cuvette 4: 0.08725
- Cuvette 5: 0.09347
- Cuvette 6: 0.15826
- Cuvette 7: 0.20356
- Cuvette 8: 0.21735
So what does this say....
Absorbance of components:
- MTs = Cuv 1 = 0.01406
- 1mM ATP = Cuv 2 - Cuv 1 = 0.0528
- 2mM ATP = Cuv 3 - Cuv 1 = 0.0535
- 3mM ATP = Cuv 4 - Cuv 1 = 0.0732
- Kinesin = Cuv 5 - Cuv 1 = 0.0794
- Reaction 1 = Cuv 6 - (MTs + 1mM ATP) - Kinesin = 0.012
- Reaction 2 = Cuv 7 - (MTs + 2mM ATP) - Kinesin= 0.0566
- Reaction 3 = Cuv 8 - (MTs + 3mM ATP) - Kinesin = 0.0507
Comparing the values of the absorbance of the three reactions to the values obtained in the standard curve, we find that...
- MTs + Kinesin + 1mM ATP ~ 18uM Pi
- MTs + Kinesin + 2mM ATP ~ 25uM Pi
- MTs + Kinesin + 1mM ATP ~ 25uM Pi
So the kinesin works! Maybe not as active as we want, but it is clearly doing something.