extreme conditions to induce protein? - im ready to do anything (Jun/15/2006 )

ok i have decided to try again to express my protein under extreme conditions....will try 18 degrees for 16 hours....was wondering did anyone try no shaking at RT overnight?? ...what other extreme conditions to solve solubility/degradation problem have you tried??? thanx a lot

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Hi Kathy, sorry for the delay. I added the high IPTG concentration at optimum OD of 0.65 and let it shake for 15mins more at 37º; then transferred it to a regulable incubator pre-set at 18ºC, and let them grow O/N (more or less 16h). I recomend you to first try a small scale culture and just analyze the resulting soluble and insoluble fractions by Coomasie (I use a standard procedure), to check that the restrictive culture conditions actually do the work with your construct.

Good luck and sorry again for the delay

miguelon

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Miquelon! thank you so much! i will try your conditions....i hope degradation (which what worries me most) will be decreased.....and im sorry im worrying everyone with my troubles...

i have heard once that some poeple leave their bacteria growing overight on the bench... have anyone tried that one?

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