Hello, I am currently trying to optimise a bacterial expression system using BL2 1DE3 bacteria and IPTG. In order to just have a quick look, whether I can see any protein overexpression on a Coomassie stain, I was wondering: can I lyse my bacterial pellets in 4x Laemmli buffer (+5% beta mercaptoethanol) for a whole cell lysate and then directly run this on a gel?
Dear Anna,
You can collect the amount of bacterial culture that corresponds to 1 ml of OD600 = 1 for each condition that you are checking, centrifuge cells for 2 min at 8000 rpm, discrard all the supernatant media and resuspend the cell pellet in 200 ul of 1x SDS-buffer (Lammli buffer). These probes you can store at -20C. Before running the gel, boil them for 5 min at 99c and then centrifuge for 5 min at maximal speed at the centrifuge (~13000 rpm), then you can load 10ul of each probe on the gel, it should be enough to see the overexpression of the protein.
Good luck!
Yes. I routinely do this to check for over-expression. 60ul of culture + 20ul of 4x sample buffer + reducing agent. I heat 5 minutes at 80C then vortex for 30 seconds to break the DNA and reduce viscosity. Load 10-20ul on PAGE. Then detect with coomassie blue
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