I have been trying to express a protein, but I won't get success. My conditions for expression were:
C3030 bacteria
LB media + kan (50ug/mL)
O.D = 0.4-0.5
IPTG 0.4mM -0.5mM
37C for 4 hours or 16C overnight, 250rpm in both
Bacteria lysis with Tris 20mM + Nacl 200mM. The bacteria were sonicated in 300W for 5 minutes.
I cannot see any protein.
Does someone have an idea what it happens?
Thank you!
I agree with Sebastian Schmitt
that you first need to be certain that your construct is ok through sequencing. Secondly sometimes you just get a bad transformant, I would retransform and pick 8-10 fresh colonies and do small scale expression and run a gel to see if any of them give you the protein band you are looking for. Be sure you solubilize all the protein with SDS buffer. It may also be that your protein is in inclusion bodies and if you sonicate and discard the pellet then you may have discarded the protein band.
Dear all, thanks for your attention. I will use a new IPTG, retransform my bacteria and redo the expression. Probably this protein will be in inclusion body solution. When I finish, I will send feedback for you.
Thanks a lot!
Dear,
I have tried to express as you recommended but doesn't work. Probably the plasmid is not good, or the bacteria is not competent.
Thanks a lot!
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