I want to digest my alpha synulein protein (monomers and fibrils). After disaggregation of the fibrils and drying via speedvac, I tried resolubilizing the fibril sample with 20ul 8M Urea,100mMTris, pH8.5. The pellet will not budge. I have tried adding more urea, vortexing vigorously, and sonication.
Any suggestions on how to proceed?
Fibrils are insoluble by their nature maybe you can disperse it with more agitation. But if your aim is to disaggregate fibirls, SDS can be definitely helpful.
HI,
As far I can understand the issue is probably due to speedvac drying which aggregates the proteins. Try to reduce the time for speedvac or use alternate means.
Additionally you can also try using guanidine hydrochloride (6M) or SDS as suggested by Amir
Hope it helps
Santosh
Hi,
Let me see if I get it right. Is that the protocol sequence that you use:
digestion assay with fibrils -> speedvac -> disaggregation of the remaining fibrils into monomers.
If yes then the coming answer might be useful. HFIP (Hexafluoroisopropanol) might do the job as well. In general you should look at compounds that break hydrophobic interactions. After the digestion assay, the remaining fibrils are even more prone to clustering (you expose a lot of hydrophobic patches after 'peeling off' all the N- and C-termini parts that do not participate in the cross-beta sheet core and are accessible for the enzyme). Probably that is the reason why your pellet holds together. Avoiding the speedvac or any centrifugation step that presses the fibrils together might help. Perhaps dialysis? Good luck.
P.S. Make sure you use the right concentration range if you go for the SDS option proposed by the other colleagues. As far as I remember, reports claim that low SDS concentration could even promote aggregation of aS into fibrils.
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