I've tried growing condition at 4c for overnight, 16c for 4 hours and 37 c for 2 hours after IPTG. Still i could not get expressed protein in soluble form. If there any more conditions?
Hi there,
Do you spot your protein as an insoluble protein or is it not expressed at all? If the former it's a matter of optimizing expression conditions, if the latter the first control is to check plasmid equence. About the conditions you have already tested, 4°C overnight is useless as there would be no expression at all. I would suggest repeating induction at 16°C overnight instead. An other parameter you might consider is adjusting IPTG concentration. Last but not least, autoinduction might also be considered.
There are several things that I would check over:
1. If you transform your cells with an empty vector do you get expression of the Maltose binding protein (MBP)?
2. What is the strain you are using? Does it have an inducible T7 RNA polymerase in the genome?
3.Check total cell extract for protein compared to a non induced sample that you have treated the same.
4. If the protein cloned in frame with the MBP tag then you can always western plot and probe against MBP, if the expression level is very low.
What concentration of IPTG are you using and when are you adding it?
Dear Dominique Liger, i have found in insoluble form bu not in soluble form. I'm using 0.1 M IPTG concentration (0.5ml/100ml solution),Thank you for your advice i will try in this condition also.
Dear Simon Cass,
Yes, i found expression of only MBP, I am using BL21 starin and i am using 0.1 M IPTG concentration (0.5ml/100ml solution). Thank you for your response i will try.
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