Hi all,
Recently I am working on protein purification.
1. affinity column(GST)
2. Add PSP enzyme to remove GST-tag(O/N)
3. GST-tag off
4. gel filtration
However, the protein tends to form large amount of soluble aggregate shown as the gel filtration figure (first peak). I need the native proteins for futher assay so how can I avoid such a problem?
You may be able to reduce the amount of aggregation by including a nonionic or zwitterionic, non-denaturing detergent to overcome hydrophobic interactions. The detergent should be included in the lysis buffer and maintained in all solutions used during purification.
Hi Harry, You can try few things.
1)Keep your protein in reducing agent at all times. You can either use 2mM DTT or 5mM BMe.
2)As Adam mentioned, use detergent. CHAPS usually do a good job for me. I usually use 0.25% in lysis buffer and maintain 0.05% throughout purification.
3) Try to use nanodrop to check 260/280 for both the peaks. if the first peak shows nucleic acid contamination and the second does not then there is a possibility your protein is bound to nucleic acids. in that case. try using benzonase in lysis buffer(do it after sonication) and incubate on ice for 30 mins to 1 hour.
Hopefully this might improve your purification a bit. orelse you might will need to try purifying it with its binding partner.
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