Inclusion Bodies in E. coli

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Feb 7 05:53:11 EST 1996


In article <4f87ls$h7t at harpo.uccs.edu>, "S. Riley"
<spriley at eclat.uccs.edu> writes
>Also after isolation of another enzyme (GSA-AT) from a size filtration 
>column in a trycine buffer, pH 7.9, we run it over a DEAE column using 
>the follwing procedure: 0-40% NaCl over 192 mls (the size of our fraction 
>collector). Unfortunately we cannot find any activity after the DEAE and 
>are not sure what has happened to our protein. If you know of any good 
>DEAE procedures please let me know.


0-40% NaCl is a little large for a DEAE gradient. Do you really mean 40%
NaCl ie 40g/100ml = 6.8M !! I would run a 0-0.5M NaCl gradient and then
a short 1M step. A gradient size of 10x column volume is typical. Most
proteins that bind to DEAE elute before 0.4M. Are you sure your protein
bound? Does it require a buffer with say 10% glyceol present or 10mM B-
EtSH present etc. Alternatively shake say 500ul of DEAE in an eppendorf
with your protein, wash and step elute with 0.1M increasing NaCl
aliquots.

Good luck

Duncan  

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