Protien expression - concentration of IPTG (Feb/08/2005 )

Hi,
I am trying to express protien, with induction of IPTG( 1mM) for 4 hours, but the expression is low. So, can I try increasing the con. What could be highest concentration of IPTG I can use??

thanks,
shilpa.

I don't think the concentration of IPTG, when using 1mM, matters for increasing the expression.
Instead, try to induce at higher density (OD600 > 0.6), or induce for longer time (overnight at 30 degrees). Or use a different strain. Some strains are optimized for expression of mammalian proteins.

from your mesage I understand that you are adding IPTG during inocluation itself. that's wrong. The correct way is to grow the cells to a mid log phase , say for 3 hrs or so and then add IPTG. 1mM IPTG is the highest concentration good enough.

If IPTG is aded in the beginning itself then the cells start expressing the protein immediately and most of the cellular resources are siphoned off and the cell division machinery is deprived.

good luck

Sharath.B
Research Fellow
National Chemical Laboratory
India

I agree with Sharath... IPTG should be added only when your bacterias are in the exponential phase of growth.

Simon