if NO expression, it may mean that your tag is useless for your target protein. Some of the most useful(i.e. expression-enhancing) tags are SUMO, NusA, MBP and GST. Also tag suitability depends on temperature range which has been recently demonstrated in a proteomic expression screen (PMID: 18226205 [PubMed] ). I do the initial screening only with autoinduction @"tag-reasonable temperature". Much simpler : 1 time series per construct. Once you have good (=solubly expressing) constructs you can still go back to (in some cases faster or more economic) IPTG-based expression. cheers, David
2010/1/8 Kyle Legate <[email protected]>: > 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. > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 (magic:) android +31 652614443 Science is what happens while we are making other plans (~John Lennon) _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
