Another Jake, on May 6 2010, 12:22 PM, said:
0.1-0.5% NP40 in your washes can help too. Also, are you certain that your "impurities" are not just degradation/cleavage products of your protein. That is VERY common in GST preps. That can alternatively be the result of codon issues when producing a eukaryotic protein in bacteria. We've recoded a few of our smaller constructs to deal with this. Otherwise, we used "codon plus" bacteria which express the tRNAs needed to avoid the problem. Both methods helped reduce the smaller bands. That solved it almost entirely for a couple of the proteins, though not all.
Also, the DTT should be added before you lyse the cells (even before sonication).
Another alternative is to tag both termini of the protein and do a double purification. That covers degradation issues and codon issues, though you lose yield.
If those are not the problem, consider a single-terminus tandem affinity purification.
edit: One other alternative is to test different induction times and temperatures. Some proteins are expressed well in a stable form in only 3 hours at 37 after induction, but some others do better at room temp for longer periods.
Thank you so much
Yes,I transformed the vector to Rosetta to promote expression, because there seems to be no expression in BL21.
I doubted the possibility of degradation, and added 6-his tag on c-terminal. But i was disappointed after eluting from Ni-NTA, because there was also several bands on SDS-PAGE, then, I had no interest to going on to next step. It's my fault. I will pick up this line again to look for improvement.
Could you tell me the role of NP40, because i have no idea about it