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?
This is a rather dated post, so I do not know if you have found a solution to your difficulties already - but there is an antibody that is specific to fibrullar alpha-synuclein available from Covance (https://store.crpinc.com), which has been tested in IHC and ELISA that could be useful as a control for your experiments (as an independent measure of fibril formation from Thioflavin S). If you have any questions about this product, or any of the other alpha-synuclein antibodies that could help you in your research - feel free to contact email@example.com and we would be happy to help you.
One question that may be especailly relevant is if you have removed the GST tag from the alpha-synuclein prior to attempting to form fibrils? GST forms dimers and could be blocking the ability of the synuclein to aggregate. Also, synuclein aggregation has been suggested to result/begin from the unstructured monomeric form of alpha-synuclein - and GST can help promote folding and theoretically could be assisting the alpha-synuclein it is attached to form the alpha-helical conformation it is postualted to adopt when membrane bound.