I am studying the glycation mediated amyloid fibril formation. When there is native protein + fibrils i am able to see the bands in Tris-Tricine SDS PAGE. But, when i precipitate the fibrils, by centrifuge (12000 RPM, 30 mins), i was not able to see the band of fibrils. I tried even native page. What could be the reason? Many thanks for your suggestions.
May be, your target protein has not been precipitated well or precipitation is very less.
LC tissue (named as No.6) has Amyloid beta A4 protein/Alzheimer's disease amyloid A4 protein/PN-II at 0.25 μg/mg tissue protein. This protein comes to membrane fraction. Membrane has been precipitated by 100,000 x g, for 90 min (ultra-centrifugation).
Are you sure that your sample buffer can dissolve the fibrils? Otherwise, you'd be only able to see the native protein in electrophoresis. Amyloid is formed by interleaved proteins in beta-helices (see for example PDB-code 5o3t), to dissolve those is not trivial.
Thank you all for your suggestions. I hope my fibrils have been adequately precipitated. I have checked AFM, TEM and UV-visible Spec. I am not sure that sample buffer can dissolve my fibril. I hope that could be the problem like Dr. Buxbaum pointed out. I tried using them without heating the sample buffer. But no results. I read somewhere, suggestion to use 8M urea in the sample buffer. May I ask you now, will that be a suitable idea? If there any other methods to dissolve the fibrils, making it suitable for electrophoresis, Please suggest. Thank you.
Electrophoresis of SDS-resistant amyloid proteins is described for example in doi:10.1016/S0076-6879(06)12003-0 and a video on Article Screening for Amyloid Aggregation by Semi-Denaturing Deterge...
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