User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/25/Assay with BGB and κ casein
Today, I decided to try the assay with all new parts: newly prepared BRB80 and buffers, BGB casein (from 6/24), the new κ casein, and a known amount of kinesin straight from the freezer. I did two assays, one with κ casein and one with BGB casein, with the hope of seeing some difference between the two casein.
I made a motility solution for each assay with rhodamine tubulin (one aliquot split between the two).
- 85uL BRB80CT (BRB80+0.2 mg/ml casein+10uM taxol)
- 1uL MgATP (at 100mM)
- 1uL dextrose (at 2M)
- 2.5uL antifade
- 10uL microtubules (one aliquot with 98uL BRB80T added after polymerization)
The only difference between the two motility solutions is using the BRB80CT with κ casein (for the κ assay) and BGB casein (for the BGB assay).
I pulled out one aliquot of the kinesin that was prepared on 6/19.
The volume in the aliquot (1uL) was too small to pipette out using our tips, so I cheated just a little bit and added 1uL of BRB80 to the aliquot and mixed it. With a total volume of 2 uL, I was able to pull out 0.2uL for each assay.
In an effort to conserve resources, I cheated even more by putting the remaining kinesin (now diluted to 2.5 mg/mL and stored in 1/2 BRB80 and 1/2 in the proper solution, 1.6 uL total volume) back into the -80 freezer. Kinesin is not supposed to be stored at this concentration or in just BRB80, but with any luck it will be alright. Trying to save it is better than just tossing it.
To each bit of kinesin, I added 250 uL of BRB80CA, and have a final concentration of 2ug/mL. I was trying to go as quickly as possible so that the kinesin would go bad, and in my haste made a calculation error and ended up adding about 2.5 times more BRB80CA than necessary. We want a concentration of between "0.05 - 5 ug/mL", and even with the mistake we should still be in range.
I put the unused portion of diluted kinesin into the -20C.
Once again, the assay did not pan out, I saw almost no signs of directed motion. In both cases though the microtubules seemed to be pretty healthy and fairly long. I did witness a few microtubules break, but it was definitely not a large enough portion of the sample to worry. The antifade also seemed to be working quite well also, with the room lights off I could see the sample clearly for several minutes.
In both cases there were a significantly fewer number of microtubules that were stick to the coverglass (a few with one end attached, but almost none were entirely stuck). This lets us know that both types of casein are working (and perhaps the older mixture lost potency).
In terms of motion of microtubles, it was rather difficult to tell the difference between the assays. There was a lot of brownian motion, however neither assay appeared to have directed motion.
The only discernible difference was in clarity. The κ casein looked much clearer (tubes against a dark background, clear distinctions). The BGB casein looked a little hazy. There were no viable chunks of casein, but it seems like it has slight opaque quality. But, as this is based on just the one test, I can not be definite.