I've been having issues with alpha synuclein I recently expressed and purified. I use the pGEX4T-1 vector and my protein is GST tagged. I induce the cultures at around an OD of 0.5-0.7. We perform aggregation assays and we use Thioflavin S binding assay to detect the beta structures in the protein. For some reason, I do not see positive Th-S fluorescence counts even after aggregating my protein for 4-5 days. The signals are very low and show no significant increase. Did something go wrong during the purification process? My SDS gel is clean and the concentration yield was around 1mg/ml. My colleague has used the same batch of BL21 cells that I used and faced no such problems. Please help. My PI is not all that happy about this situation.
I have just recently expressed and purified alpha synuclein from BL-21 DE3 cells. The issue is, my protein won't aggregate even after 5 days of shaking at 37C. Now I can't think of reasons why this is happening. My Th-S counts have abrely changed in the last 5 days. It is slightly bizarre and we have never had this problem before. Has anyone had similar problems and what have you done to solve this?