User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/16/Motility Assay: Attempt
Today we made our first attempt at making an inverted motility assay. In the assay we want to coat our surface with casein, add kinesin (which will bind the the casein) and then add microtubules which will be kicked along in a directed motion by the kinesin. The directions for this assay were taken from a printout, I have no idea who originally did the assay from which I am copying (perhaps Koch?), but hopefully this counts as a citation. Here is the run down on how to make this assay.
Making Various Buffers
I made 0.5mL (500uL) of each of the following buffers, but 1mL of BRB80T.
- BRB80T: BRB80 + 10uM taxol
I used 990 uL of BRB80 and 10 uL of taxol (at 2 mg/mL)
- BRB80-CT : BRB80T + 0.2 mg/ml casein
I used 450 BRB80T (made from the recipe above) and 50 uL of 2 mg/ml casein
- BRB80CA: BRB80 +0.2 mg/ml casein + 1mM MgATP
(50 uL of 2mg/mL casein + 5 uL of 100mM MgATP + 445 uL ofBRB80)
- BRB80CS0.5: BRB80 + 0.5 mg/mL casein
(100 uL 2.0 mg/mL casein + 400 uL BRB80)
Motility Solution
Recipe:
- 85 uL BRB80CT
- 1uL MgATP (100mM) 1mM
- 1 uL D-glucose (2M) (aka, dextrose) 20 mM
- 1 ul glucose oxidase (2 mg/mL) (GOD) 0.02 mg/mL
- 1 uL catalase (0.8 mg/mL) (CAT) 0.008 mg/mL
- 0.5 uL BME 0.5%
- 10uL MT100 (0.32 uM) ~ 32 nM
- Total: 99.5 uL
GOD+CAT+BME in these ratios and concentrations is indentical to the antifade solution that was already mixed up, so I added 2.5 uL of the antifade in place for the GOD, CAT, and BME.
Protocol
Create a flow cell on a slide and coverglass using double stick tape. Flow in: 20 uL BRB80CS0.5, then wait for 5 minutes 20 uL kinesin (0.05 - 5ug/mL, dilute in BRB80CA), wait 5 minutes 20 uL motility solution Wick these solutions with kimwipes
We made two cells, one that had just the motility solution and one that had the entire assay. Koch mentioned that the kinesin was very old (from 2004) and had been stored in the -20C freezer instead of the -80C (as it was supposed to). Also, it appeared to have about 10 times the concentration as required by this assay, so I diluted 5uL of kinesin with 45 mL of BRB80CA).
Under the microscope the mixture of just the motility solution seemed alright. The microtubules were not entirely stuck to the glass, if they were it was just at one end.
The assay was very much the same story, unfortunately. The microtubules were moving, but with Brownian motion, not the directed walking that we were hoping for.
Try #2
Koch proposed that a reason for this could be that the kinesin was very old and had perhaps lost a lot of of its potency. We made a new slide with 10 times the concentration of kinesin (pipetted straight out of the old stock). I then added 1uL of casein to the 20 uL of kinesin (since we are not adding BRB80CA, casein needs to be added independently).
This assay also failed. It looked nearly identical to the first try, the only difference being that slightly less microtubules had stuck to the glass.
I think that a major reason for this assay not working was the age and storage of the kinesin. I think the next logical step in getting this assay working is to try it again with newly prepared kinesin.
