janu Posted March 21, 2008 Share Posted March 21, 2008 hi all i have cloned 600 bp gene in pet vector in colony pcr i have got positive result ,i have got amplification in that once after expression analysis of protein with IPTG induction i am getting very less amount of expression can anybody suggest me how to improve expression of target recombinant protein thanks in advance Link to comment Share on other sites More sharing options...
ecoli Posted March 21, 2008 Share Posted March 21, 2008 without knowing anything more about your protein or strain, I would say try inducing for longer or maybe using a higher concentration of IPTG. Try a bunch of different conditions and run then on an SDS-PAGE gel simultaneously. Link to comment Share on other sites More sharing options...
janu Posted March 27, 2008 Author Share Posted March 27, 2008 protein is human growth hormone and strain is e coli bl21 Link to comment Share on other sites More sharing options...
CharonY Posted March 27, 2008 Share Posted March 27, 2008 Unfortunately there are a gazillion possibilities why it does not work. First question is whether there is production of the correct protein at all. Based on the OP I'd assume that there was low production. Have you checked whether it is the correct protein (e.g. by MS/MS)? Link to comment Share on other sites More sharing options...
ecoli Posted March 27, 2008 Share Posted March 27, 2008 what vector? One time, I spent 6 months trying to successfully clone my insert into a pMMB expression vector. Link to comment Share on other sites More sharing options...
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