Two-Dimensional Semi-Denaturing Agarose Gel Electrophoresis: A Technique to Separate Polymorphic Amyloids Fibers Based on Size Heterogeneity

1. Prepare Samples

1. Culture 2 x 10⁶ amyloid producing HT-29 colon cancer cells in a 10-cm tissue culture dish in 10 mL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and penicillin-streptomycin. Culture cells overnight in a 37 °C incubator with 5% CO₂.
   1. After the cells grow to 80% confluency, wash the cells with 10 mL of phosphate-buffered saline (PBS). Add 3 mL of Trypsin solution and incubate at 37 °C for 3 min.
   2. After the cells are totally dissociated from the dish, add 10 mL of culture medium and transfer the cells with a 10-mL pipette to a 15-mL conical tube. Centrifuge the cells at 1,000 x g for 3 min at room temperature. Aspirate the medium, resuspend the cells in 5 mL of culture medium, and count the cells using a cell counter. Plate 2 x 10⁶ cells in each of two 10-cm dishes.
   3. Allow the cells to adhere and recover overnight in a 37 °C incubator with 5% CO₂. Apply treatment to one dish to induce the formation of amyloids with 20 ng/mL Tumor Necrosis Factor-Alpha (TNF-α), 100 nM Smac-mimetic, and 20 µM pan-caspase inhibitor Z-VAD-FMK. The combination is abbreviated as TSZ. Treat the other dish with vehicle as a control.
2. After the appropriate length of time, usually 6 h, harvest the cell lysate.
   1. Scrape the cells off the plate with a plastic scraper and use a 10-mL pipette to transfer into a 15-mL conical tube. Centrifuge the cells at 1,000 x g for 3 min at 4 °C.
   2. Wash the cells 2 times by resuspending in 10 mL of ice cold PBS and centrifuging at 1,000 x g for 3 min at 4 °C. Aspirate the PBS solution.
      NOTE: The process can be paused here by freezing the cell pellet in liquid nitrogen and storing at ˗80 °C for up to 1 month.
   3. Transfer the cell pellet to a 1.5-mL microcentrifuge tube and incubate in 0.3 mL of lysis buffer for 30 min on ice. Centrifuge at 20,000 x g for 15 min at 4 °C. The supernatant is the whole cell lysate.
      NOTE: The process can be paused here by storing the sample at -20 °C for up to several months.
   4. Measure the protein concentration by a Bradford assay. Add 4x SDD-AGE loading buffer to prepare 20 µL of 3 µg/µL sample and incubate at room temperature for 10 min.

2. Prepare and Run Gels

1. Add 2 g of agarose to 200 mL of 1x Tris-acetate buffer (TAE) in a glass beaker and heat in a microwave to melt the agarose. Add 1 mL of 20% SDS for a final concentration of 0.1% SDS. Carefully swirl to mix. Take care not to generate bubbles after the SDS addition.
2. Pour the agarose solution into a 15 cm x 14 cm gel slab. Use a 1-mL pipette to eliminate any bubbles. Place one 20-well comb at the top.
3. First dimension: Pipette 60 µg of whole cell lysate in the far-right lane. Run the gel at 60 V for about 4 h (until the dye front is about ¾ through the gel) using the TAE containing 0.1% SDS as the running buffer.
4. Second dimension: Carefully rotate the gel 90° counter-clockwise (Figure 1A). Run the gel at 60 V for about 4 h.
   NOTE: The general running condition is 4 V/cm gel length. It is important that the running conditions are exactly the same for the first and second dimensions.