Protein expression and purification - Please help - (Apr/29/2013 )

Hey there,

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?

Hi salema,

            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 ab.products@covance.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.

-Chris-