If your protein is a membrane protein, you wouldn't necessarily expect it to remain soluble after lysis and centrifugation. If it works with your experimental needs, I'd recommend re-cloning your protein into your plasmid without the transmembrane domains. Alternatively, you can try resolubilizing your protein with detergents. There's also a chance that some of your protein might be soluble still. After centrifuging and clarifying your lysate, you can try to ultracentrifuge your sample. This should pellet any soluble membrane species that might contain your protein of interest. Expressing membrane proteins in E. coli is hard and there aren't any easy answers that will always work.

Yes some of my protein is soluble, but very low. It is as if I don't do induction.

So may I ask the details of resolubilization of the protein in detergent option ? To the pellet of the culture or to the debris after lysis I need to add detergent you mean ? I tried adding different detergents both separately and together in my lysis bufffer but it was not good enough..

Thank you !

Hi Sinem, is your protein in inclusion bodies? If so, you can spend forever and a year to try to solubilize it, but it will never behave like the native protein.

My advice is to harvest the cells before your proteins get aggregated into inclusion bodies, which are like the cells garbage disposal unit with a mess of misfolded and insoluble proteins.

I tried solubilizing a membrane protein that I was expressing in E. coli quite a few years ago. From what I remember, it is usually best to treat the pellet after clarification with a detergent solution; doing so helps to purify your sample. From what I remember, you may want to try using a wide range of detergent concentrations (from the CMC up to 10% solutions). I should qualify my statement by saying that I never succeeded in purifying the membrane protein I was working on and wound up switching to a different project in the lab.

I don't think it is in inclusion bodies, because I can not open it with urea I would say..

Thank you so much! I will try adding detergent to the debris after clearance of the lysate then, maybe it works for me...

Hi Sinem, if you need denaturing agents to solubilize your expressed protein then it a sure sign that it is in inclusion bodies.

You can try a simple experiment. Induce a batch of cells and take a sample and lyse after 1hr, 2hr, 4hr, 6hr and overnight incubation.

Do a mini Talon (or whatever resin you use) capture assay to assess His binding.

Put 100ul of resin in a tube + 1ml of lysed cells. Incubate, wash and boil the resin pellet in SDS sample buffer and run on a gel. Blot and probe with Ab against your protein to see what conditions are best for capturing your (hopefully soluble) protein.

Thank you so much..

I wonder some other thing as well. Do you think I can induce my cells at 4 degree Celcius.. I decreased the temperature until 26 degree and either 18 or 4 degree I am thinking of trying, although I don't know how much logic it is..

Hi Sinem, I think you have to induce the cells when they are close to their peak growth and peak biosynthesis.

If you can confirm that the problem in purifying your protein is due to its deposition into inclusion bodies, then you have a clear map of how to optimize induction, growth time, harvest and how to bust the cells without destroying your protein.