After several rounds expressing labeled proteins I realize that the yield of the expression is lower that I expected, in fact I'm afraid of the final protein concentration is lower than what I will need for the further experiments. How can I improve the yield of the expression using this media? What is the best protocol of the M9 minimal media?
I have seen people first grow their cells in LB media to the desired density and then pellet the cells with gentle centrifugation. After centrifuging, the cells are resuspended in the minimal media and then induced. It may also help to grow the cells to a higher OD than usual before induction. I typically induce at an OD over 1.0 because my cells stop growing entirely after induction. I hope this helps!
Jacob
Compared to LB, you'll get about half the maximum cell density with M9 (2g/L glucose), so expect less protein vs. the equivalent volume of LB. With that said, to maximize expression in M9, here are a few things we try:
1) In addition to Jacob's suggestion to induce late, also induce early (actually, do an induction screen, 0.5-1.0 OD. Some of my proteins needed to be induced at 0.9, some didn't care.
2) Try different cells. I start with the simple cells - BL21(DE3)-Tuner and get more complicated - C41, Rosettagami, Codon+RIPL, etc. as necessary. As long as you're making your own competent cells, this is essentially free. Again, some constructs are very sensitive, some are not.
3) Screen induction times & temperatures. My default is now to grow @ 37 / induce overnight @ 15 (also means you aren't inducing cells at 4PM and having to harvest 3 hours later). But I had one protein that was grow @ 32 and induce @ 22.
4) Reclone into a different vector. I have a set of different T7-driven vectors with different fusions and the same restriction sites and usually drop my insert into 2-3 of these for a grow-off. Maltose-binding protein usually (but not always) wins.
5) Silly things - I still add B-vitamins, preferably less than a year old (I've read that newer strains don't need these, but historical contingency wins); I never make stocks of IPTG - I mix it up while the cells are growing.
The rules change a bit if you're growing in D2O, but even there my principle variant is getting the cultures started, not yield, so test the various conditions in unlabeled M9.
-Dave
A couple of things which have helped here:
1) Make sure that you always work from freshly transformed cells.
2) Grow the cells to a higher level than the equivalent on LB prior to induction.
3) If all else fails it can sometimes be worth supplementing the M9 with a small aliquot of labelled rich media if your protocol allows.
Phil
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