Inclusion Bodies in E. coli

Dr. Duncan Clark duncan at
Wed Feb 7 05:53:11 EST 1996

In article <4f87ls$h7t at>, "S. Riley"
<spriley at> 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

Good luck


The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

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