How can I get protein in soluble fration
Posted 11 September 2010 - 02:47 AM
I have cloned tow fragments ( 30 and 24 KDa)from the same protein (human)into 3 different vetors ( pGEX, pMAL and pHUE) with 3 different tags ( GST, MBP and HIS6-Ubiquitin tags). I need to purify them in native condition. I did a trail expression in E. coli ( BL21 and Rosseta) at 37C and 20 degree with 0.4 mM IPTG. In all cases, all the six construts were in an insoluble fraction and almost nothing is in the supernatant.
Could you please help me to make these proteins soluble .
Posted 11 September 2010 - 09:58 AM
I am not sure if this would help but I know that some proteins, in particular certain enzymes, can be solubilised using high concentrations of Urea (i.e. 4-6 M Urea). In this way you can get them out of tissues in their native form. Still, you will need to purify your protein afterwards using dialysis.
Posted 12 September 2010 - 09:53 PM