This article discusses the analysis of amyloid fibers using two-dimensional semi-denaturing agarose gel electrophoresis. It highlights the method's ability to separate intact fibers based on size, revealing their heterogeneity.
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.
To prepare the gel, add 2 grams of agarose powder to 200 milliliters of TAE buffer in a glass beaker. Then, heat the beaker in a microwave to melt the agarose.
Next, add 1 milliliter of 20% SDS to a final concentration of 0.1%. Gently swirl the beaker.
Next, pour the liquid agarose on a 15 cm x 14 cm gel slab. To remove any air bubbles, use a 1-milliliter pipette. Then, place a 20-well comb on top of the gel.
Next, add approximately 60 micrograms of whole-cell lysate in the far-right lane of the gel. Run the gel at 60 volts for about four hours, using the TAE buffer containing 0.1% SDS, as the running buffer.
To run the gel in the second dimension, carefully rotate the gel by 90 degrees counter-clockwise. Then, run the jail at 60 volts for about four hours.
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