Hello, recently I've been trying to synthesize the α-synuclein fibrils by purchasing the commercial α-synuclein proteins (BostonBiochem Recombinant Human His6 Synuclein-α Cat. # SP480).
I have used the following method to make α-synuclein fibrils by reference (van Raaij, M. E. et al. 2006. Biophys. J. 91:L96-L98.).
In briefly, the α-synuclein solution is diluted with 10 mM HEPES buffer, 50 mM NaCl, pH 7.4. The final concentration of α-synuclein solution is 25 μM. The α-synuclein solution is distributed with 1 ml in the microtube and then incubated in the oil bath for 6 days under constant stirring 300 rpm. However, we could not find fibril in AFM imaging and I don’t know why. For your information, we did not purify the purchased protein.
There are four things we consider to be the cause of the problem.
Concentration (Is this concentration too low?)
Purification (Should I purify the purchased protein?)
Buffer solution (any recommend?)
His6 tagged protein (Is it matter?)
Is there anybody who experienced the same situation? Please recommend any method to synthesize fibrils in vitro.
I'll wait for your comments! Thanks!
His-tagged syn is not a frequent one in papers. I attached your paper's Supp (mmc1.pdf), where the preparation was described for untagged syn.
The bast way to do this is to use a protocol that takes the reaction all the way to fibrillar form, and then sonicate. There are many ways to do this, but the key is letting the reaction go long enough. In my biased opinion, Ronald Melki is doing the best work in this area, I advise you to read this paper....Good Luck
Article Structural and functional characterization of two alpha-synu...
Hi Wonseok,
If you're still working on this and looking to save some time, you can purchase alpha synuclein fibrils here: https://www.stressmarq.com/products/protein/alpha-synuclein-protein-spr-322/
Good luck!
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