Protein expression and purification - Please help - (Apr/29/2013 )
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.
how much do you load? do you have controls?