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pGEX-2t purification problem


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#1 seeking_2012

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Posted 01 September 2013 - 12:22 PM

I have my gene cloned into pGEX-2t , I changed everything temp, concentration IPTG, Combatant cells but protein still insoluble.
I do not know if using Urea can destroy my protein or not cause I will do methyltransferase assy in this protein. Is using L- Arginine with the medium that will help solubleization?. I am wondring if anyone used this before .
Many thanks for  you help



#2 bob1

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Posted 03 September 2013 - 02:32 AM

Which lysis buffers have you tried?  Detergents? Salt concentrations? denaturing agents? glycerol?



#3 seeking_2012

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Posted 03 September 2013 - 03:46 AM

I have used PBS and sonication then I add triton 1%



#4 bob1

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Posted 03 September 2013 - 12:36 PM

Try a range of salt concentrations from 500 mM to 100 mM, addition of 2-ME and glycerol can help sometimes, as can trying different detergents.






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