Biology 2382B Lecture Notes - Lecture 9: Microtubule
5 May 2018
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How Do We Find the Proteins that Transport Materials Along Microtubule?
1. Inject radioactive AA into axon of living animal (ex. squid axon)
2. Radioactive AA incorporated into proteins and the radioactive proteins are transported into axon
3. Wait 5 minutes
4. Cut axon into distinct fragment relative to injection site (1 cm, 2 cm, 3 cm, and 4 cm from site).
5. Isolate proteins from fragment and look for the radioactive proteins if there were proteins that
were made next to the nerve fragments, and proteins are radioactive, they can be run on SDS gel to be
separated by size. Then, you can see the radioactive protein present by putting gel on X-ray.
- area right next to injection site has lots of proteins, area 2 cm from injection site there are less
proteins, and area 3+4 cm from injection site have no proteins.
- run the experiment again (same injection in same location) but wait 10 min (then 15, 20, etc.) instead
of 5 min, and isolate the same nerve fragments (1,2,3,4 cm from injection site) and do the same thing.
You’ll ed up seeig the sae patter.
Result:
i) Proteins travel and work together in GROUPS (groups = motor complexes)
ii) Different bands represent proteins travelling at different speeds in cells moving @ defined rate
away from injection site, forward (not diffusion)
- this is how kinesins were discovered
find more resources at oneclass.com
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Document Summary
Then, you can see the radioactive protein present by putting gel on x-ray. Area right next to injection site has lots of proteins, area 2 cm from injection site there are less proteins, and area 3+4 cm from injection site have no proteins. Run the experiment again (same injection in same location) but wait 10 min (then 15, 20, etc. ) instead of 5 min, and isolate the same nerve fragments (1,2,3,4 cm from injection site) and do the same thing. Result: proteins travel and work together in groups (groups = motor complexes, different bands represent proteins travelling at different speeds in cells moving @ defined rate away from injection site, forward (not diffusion)
