I have never had problems in over-expressing my protein in BL21 strain with IPTG 1 mM (final concentration). However, now in my stay in Portugal I can't over-express it.
I have tried differents concentrations of ITPG and ITPG from other labs. However, a labmate checked our ITPG with her conditions and protein and it is ok, her protein is over-expressed however mine isn't.
So the problem is not with the IPTG. Could be then that the problem is in my plasmid?. I checked my miniprep (plasmid pET28a + insert) and the size was ok. But I brought my samples lyophilizated. Could be that although the size is right, the plasmid could be damaged?.
I don't have any idea why I have this problem.
Alba,
It could be an issue with toxicity of your plasmid or protein. Have you tried using other cell lines such as C41 and C43? I have had some plasmids that do not over-express in BL21, but have great over-expression in C43. Also you may want to try another plasmid to see if you can get better expression. I hope this helps!
Jacob
Thank you very much for you answer. I will try to change the cell line. But it strange because I have never before had problems with BL21 and my plasmid.
Thank you for your advice!
Did you check about sequencing result? Previously I found the reason of T7 promoter has mutation .
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