Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity
Transcription
Amyloid fibers are aggregates of misfolded proteins, which exhibit size heterogeneity. These fibers, being resistant to anionic detergents such as SDS, can be analyzed using two-dimensional semi-denaturing agarose gel electrophoresis.
To begin, take a pre-assembled, large pore-sized agarose gel that contains SDS. The high porosity allows the separation of intact fibers during the run. Overlay the gel with a running buffer containing SDS.
Load a cell lysate containing amyloid fibers into the gel. The presence of SDS and the absence of sample heating step creates semi-denaturing conditions where the detergent binds to highly resistant amyloids imparting a uniform negative charge to the intact fibers.
Run the gel in the first dimension at the appropriate voltage. The electric field causes the migration of negatively charged amyloids toward the positively charged anode.
During the run, smaller fibers migrate quickly compared to larger ones – forming a smear-like band pattern indicating size heterogeneity.
Upon completion, rotate the gel perpendicularly and run it in the second dimension. In this dimension, fibers move identically to the first run separating based on their sizes. This creates a diagonal smear as smaller fibers migrate faster than the larger ones.
The formation of the sharp diagonal band confirms an intact but heterogeneous amyloid fibrillar population.
