User:Steven J. Koch/Notebook/Kochlab/2009/06/16/Motility assays: Difference between revisions
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[[User:Steven J. Koch|Steve Koch]] 16:52, 16 June 2009 (EDT): We're going to give motility assays a try. | [[User:Steven J. Koch|Steve Koch]] 16:52, 16 June 2009 (EDT): We're going to give motility assays a try. | ||
Linh's notes: http://openwetware.org/wiki/User:Linh_N_Le/Notebook/2009/06/16#kinesin | |||
==Attempt at assay== | |||
* Made a double flow chamber. Top will be just motility solution. Bottom will be full motility assay. | |||
* Incubating with BRB80CS0.5 for 5 minutes (just over) (sample volume is about 14 ul) | |||
* Incubating with kinesin (diluted 1:10 in BRB80CA), 15 ul, just over 5 minutes. | |||
* Flowed in 15 ul motility solution. Observing now. | |||
==Kinesin== | ==Kinesin== | ||
We're going to try an old aliquot of drosophila kinesin that I have from 2004. Yeah, that's really old, but hopefully it will have a little activity. I don't have a lot of details about the kinesin. So, we're just going to assume that the stock I have is 20 ul of about 1 mg / ml protein. I think for the first attempt, we should dilute this 5 microliters plus 45 ul of BRB80CA. | We're going to try an old aliquot of drosophila kinesin that I have from 2004. Yeah, that's really old, but hopefully it will have a little activity. I don't have a lot of details about the kinesin. So, we're just going to assume that the stock I have is 20 ul of about 1 mg / ml protein. I think for the first attempt, we should dilute this 5 microliters plus 45 ul of BRB80CA. | ||
* Brigette is adding 45 ul of BRB80CA to a new tube | |||
* Then adding 5 ul of "1 mg / ml" kinesin | |||
==BRB80CT== | ==BRB80CT== | ||
| Line 10: | Line 20: | ||
==BRB80CA== | ==BRB80CA== | ||
[[User:Steven J. Koch|Steve Koch]] 19:00, 16 June 2009 (EDT): I was a little worried that the[[Koch Lab:Inventory/ATP|ATP]] wasn't mixed properly. We should keep this in mind. | |||
==MTs== | ==MTs== | ||
Brigette polymerized one of the 1 ul aliquots of TRITC tubulin from yesterday. Stabilized with (approx 50 ul) of BRB80T. | Brigette polymerized one of the 1 ul aliquots of TRITC tubulin from yesterday. Stabilized with (approx 50 ul) of BRB80T. Then after making more BRB80T, she added to bring up to approximately 100 ul. Linh verified that we had tubes via microscopy w/o antifade. | ||
==Antifade== | |||
Linh is checking whether the aliquot of antifade from yesterday is OK. | |||
==[[/Need to order|Things to order]]== | ==[[/Need to order|Things to order]]== | ||
Latest revision as of 20:55, 16 June 2009
Steve Koch 16:52, 16 June 2009 (EDT): We're going to give motility assays a try.
Linh's notes: http://openwetware.org/wiki/User:Linh_N_Le/Notebook/2009/06/16#kinesin
Attempt at assay
- Made a double flow chamber. Top will be just motility solution. Bottom will be full motility assay.
- Incubating with BRB80CS0.5 for 5 minutes (just over) (sample volume is about 14 ul)
- Incubating with kinesin (diluted 1:10 in BRB80CA), 15 ul, just over 5 minutes.
- Flowed in 15 ul motility solution. Observing now.
Kinesin
We're going to try an old aliquot of drosophila kinesin that I have from 2004. Yeah, that's really old, but hopefully it will have a little activity. I don't have a lot of details about the kinesin. So, we're just going to assume that the stock I have is 20 ul of about 1 mg / ml protein. I think for the first attempt, we should dilute this 5 microliters plus 45 ul of BRB80CA.
- Brigette is adding 45 ul of BRB80CA to a new tube
- Then adding 5 ul of "1 mg / ml" kinesin
BRB80CT
Brigette made more BRB80T, using PEM from cytoskeleton. She actually made it with 20 μM taxol (as opposed to the usual 10 micromolar)--this is fine.
Brigette made BRB80CT by adding 50 μL of 2 mg/ ml casein to 450 μL of BRB80T.
BRB80CA
Steve Koch 19:00, 16 June 2009 (EDT): I was a little worried that theATP wasn't mixed properly. We should keep this in mind.
MTs
Brigette polymerized one of the 1 ul aliquots of TRITC tubulin from yesterday. Stabilized with (approx 50 ul) of BRB80T. Then after making more BRB80T, she added to bring up to approximately 100 ul. Linh verified that we had tubes via microscopy w/o antifade.
Antifade
Linh is checking whether the aliquot of antifade from yesterday is OK.
