ms wrote:
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
You're doing too much work for a simple expression screen. This is what I would do to establish some key conditions, specifically an IPTG concentration course and an induction time course (doing it at different temperatures as well will set 3 parameters):
Grow a seed culture to log phase but do not let it saturate. Set up a bunch of tubes with 0.8 ml of medium and seed each with 0.2 ml of your seed culture. Monitor one of the cultures for OD and induce when it's firmly in log phase, around 0.6-0.8. Induce with a range of IPTG concentrations (0.5 mM should be the highest concentration, I have had best results with much less), and incubate for various time points, say 1 hour, 2, 4, 6, 10, overnight. At the conclusion of each time point, spin down the whole culture in an eppi and lyse the cells in SDS load buffer. Run the whole sample on a gel. Take the best condition and scale it up.
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