ts embryo extracts

[Richard S. Jones]

We are planning on making embryo extracts from temperature-sensitive mutant 
stocks for the purpose of examining the effects of different ts alleles on 
protein-protein interactions (per co-immunoprecipitation).  Has anyone out there
made ts allele embryo extracts, or know of helpful references?  Our primary 
questions concern temperatures at which the extracts should be prepared and 
temperatures at which to do the IPs.  In order to minimize protein degradation, 
we normally try to keep everything at 4 to 0 degrees C during extract 
preparation and subsequent IP steps.  However, we are concerned that such low 
temperature incubations might permit refolding of mutant proteins and 
restoration of some interactions that are normally disrupted at restrictive 
temperature.  Is this a real problem or are we just being paranoid?  Any 
suggestions, protocols, or references would be much appreciated.

Thanks

Rick Jones
Dept. of Biological Sciences
Fondren Science Building
Southern Methodist University
Dallas, TX 75275-0376
TEL: 214-768-3810
FAX: 214-768-3955
email: rjones at mail.smu.edu