Question: you're taking a 3-ml equivalent out of 500 ml culture, and processing it as if it was a 3ml culture? Or are you basing the result on processing the entire 500ml?
Reason I ask this is simply to make sure your extraction/purification timing is the same in both cases. It matters! Assuming that the protein really is not soluble at 500ml and is somewhat soluble at 3ml - which expression set up are you using? IPTG or autoinduction? more details is better. For example, if you use IPTG - how do you determine when to induce - OD? or some other measure? The advice to change aeration is solid. Try it both ways, meaning up or down - it could go either way depending on the difference in your set up. A 3ml culture in a 24-well block at 800 rpm is MUCH better aerated than 500ml culture in non-baffled 1L flask at 250rpm. You also may want to play with temperature, and if you're not using AIM - try it out. Artem - Cosmic Cats approve of this message On Thu, Mar 19, 2015 at 2:33 PM, Reza Khayat <[email protected]> wrote: > Hi, > > > > We can express quite a bit of soluble protein when growing 3ml cultures. > However, the protein becomes insoluble (inclusion bodies) when we scale up > to 500ml cultures. Has anyone experienced such a problem, and found a > solution to it? Thanks. > > > > Best wishes, > > Reza > > > > Reza Khayat, PhD > > Assistant Professor > > Department of Chemistry > > City College of New York > > New York, NY 10031 > > http://www.khayatlab.org/ > > 212-650-6070 > > >
