Purifying axonemes

[Doug Weibel]

I recently used the method of Witman (JCB, 1978, 76, 729) to isolate and 
reactivate axonemes from C. reinhardtii and found the yield of axonemes 
purified by this method to be very low. 

I am wondering if anyone with experience purifying/reactivating axonemes 
might lend some advice.  I started with 400 mL of culture grown 
synchronously using a light/dark cycle.  Cells were clearly swimming 
before starting the procedure.  I was surprised not to see any axoneme 
pellet during the high g spins, but attributed this to isolating only a 
small amount of protein.  Perhaps these spins were too slow/short?

Any advice/troubleshooting would be most welcome.

Thanks in advance,
Doug
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