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Protein expression and purification - Please help

protein purification alpha synuclein Th-S binding assay

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#1 salema

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Posted 29 April 2013 - 06:18 AM

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.

Edited by salema, 29 April 2013 - 06:19 AM.


#2 Curtis

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Posted 30 April 2013 - 12:25 AM

how much do you load? do you have controls?

#3 CovanceABP

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Posted 20 June 2014 - 11:47 AM

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-







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