problem in protein expression

ms via methods%40net.bio.net (by monikagicts1 from gmail.com)
Fri Jan 8 04:45:11 EST 2010


hi all,
i am doing standardisation for protein expression and for this i am
taking 50 ml culture for induced and 50 ml for uninduced and earlier i
used .5mM IPTG conc. but i didnot see any expression. this i did twice
and also after IPTG induction i am keeping it at 18 degree for 12
hours and also the lysis buffer i am adding is 50mM Tris and 1mM EDTA
and i am not adding nacl but in wash buffer i am adding 150mM
NACLshould i add this in my lysis buffer and this time i am using 1mM
IPTG concentration and i am growing my culture at both 37 degrees and
18 degrees when the OD of my secondary reaches 0.4-0.5, earlier i was
inoculating secondary at the OD of 0.3.but this time i have changed
this also. actually the earlier condition i was using for the full
length but this is the same protein but with 18 residues deleted at
the c terminal so i thought i have to change the conditions also
because earlier conditions which i used didnot work .so please suggest
me so that i woulb be able to get the expression and i can start my
purification.
thanks
ms


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