How to get soluble protein?
Posted 30 April 2012 - 12:38 AM
I'm struggling with one of my protein trying to get soluble proteins. I tried lots of different constructs form that protein that predicted to be structured. I used E coli for over expression diff tags, temp, IPTG, buffer, sonication, lysozyme, commercial lysis buffer.... and the results were consistent no soluble protein. I did a couple of refolding exp but no luck to get enough stable and soluble protein.
I've heard about some Ecoli cells competent cells but I don't know if it is just take money and time for nothing. Any ideas and suggestions will be highly appreciated.
Thank you so much for your help.
Posted 30 April 2012 - 02:12 AM
Posted 30 April 2012 - 05:02 AM
Yes I tried 18 o/n, 25 o/n and 37 degree for 3-4 hours at 0.1, 0.5 and 1.0 mM IPTG in BL21 (DE3) and Reosetta2.
The size of protein from 20-45KDa +the tag GST, His or MBP.
phosphate, Tris and HEPES, 150-500 mM NaCl, DnaseI, PMSF or protease inhibitor (tablet).
I will try a detergent eventhough I tried 1% TX(100) previously.
If you send me your protocol I can try because mine never worked for me as well.
All my constructs are N-terminal tagged I was wondering if the C-termiual tag could make a big differences.
Posted 30 April 2012 - 07:41 AM