The overall goal of this two-dimensional semi-denaturing detergent agarose gel electrophoresis is to confirm size heterogeneity of amyloid or amyloid-like fibers seen during traditional SDD-AGE. This method can help answer key questions in the amyloid or protein aggregation fields, such as whether size heterogeneity seen during traditional SDD-AGE is the native state of the fibers in vivo or the result of protein degradation or dissociation during gel electrophoresis. The main advantage of this technique is it can be performed easily in lab without specialized equipment.

No additional equipment is required beyond what's used in traditional SDD-AGE. First, seed the amyloid-producing HT-29 colon cancer cells in a 10-centimeter tissue culture dish. Then, incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.

Once the cells reach 80%confluency, use phosphate-buffered saline to wash the cells. Then, add three milliliters of trypsin to the cells, and incubate at 37 degrees Celsius for three minutes. After the cells have detached from the culture dish, add 10 milliliters of culture medium to the plate.

Then, transfer the cell suspension to a 15-milliliter conical tube. Next, centrifuge the cell suspension at 1, 000 times g for three minutes at room temperature. After the centrifugation, aspirate the medium and resuspend the cell pellet in five milliliters of culture medium.

Count the cells using a cell counter. Then, leave two culture plates at 37 degrees Celsius overnight. Next, add TSZ reagents to one of the culture dishes as treatment.

After about six hours, harvest the cells using a plastic scraper. Next, transfer the cell suspension to a 15-milliliter conical tube, and centrifuge at 1, 000 times g for three minutes at four degrees Celsius. Then, wash the cell pellet twice with 10 milliliters of ice-cold phosphate-buffered saline, and repeat the centrifugation process.

Next, aspirate the PBS solution, and transfer the cell pellet to a 1.5-milliliter microcentrifuge tube. Add 0.3 milliliters of lysis buffer to the cell pellet, and incubate on ice for 30 minutes. Again, centrifuge at 20, 000 times g for 15 minutes at four degrees Celsius.

The supernatant obtained is the whole cell lysate. To the supernatant, add 4X SDD-AGE buffer to prepare 20 microliters of three micrograms per microliter of sample. Incubate the sample at room temperature for 10 minutes.

To prepare the gel, add two 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 one milliliter of 20%SDS to a final concentration of 0.1%Gently swirl the beaker.

Next, pour the liquid agarose on a 15-by-14-centimeter gel slab. To remove any air bubbles, use a one-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 counterclockwise.

Then, run the gel at 60 volts for about four hours. In a 20-by-20-centimeter container, add 500 milliliters of transfer buffer. Just immediately next to the container, prepare a five-centimeter-high stack of paper towels.

Then, soak two filter papers, each 14 by 15 centimeter dimensions, in transfer buffer, and place on top of the paper towel stack. Activate a 14-by-15-centimeter PVDF membrane in methanol for 30 seconds. After activation, place the membrane on top of the filter papers, and use a roller to remove all the bubbles.

The most important part of the transfer is to make sure that no air bubbles are formed between the gel and membrane. Rinse the gel with transfer buffer, and layer it on top of the membrane. Roll out any bubbles formed.

Next, use a plastic wrap to cover the edge of the paper towels closest to the transfer buffer container. Then, soak a piece of filter paper with 15 by 35 centimeter dimensions in the transfer buffer. Place the filter paper such that one end covers the top of the gel and the other end is in the transfer buffer container.

Cover the container with plastic wrap, and leave overnight at room temperature. The next day, rinse the membrane with 50 milliliters of PBST in a 20-by-20-centimeter container. Next, add 20 milliliters of 5%milk in PBST on the membrane.

Then, leave the membrane on the rocker at room temperature for 30 minutes to block. Pipette 10 microliters of rabbit anti-MLKL antibody into 20 milliliters of 5%milk in PBST. Then, add the antibody mix on the membrane.

Incubate the membrane on a rocker overnight at four degrees Celsius. Then, wash the membrane with 20 milliliters of PBST for five minutes. This wash is repeated five times.

Next, pipette four microliters of anti-rabbit-HRP antibody to 20 milliliters of 5%milk in PBST. Add the antibody on the membrane. Leave the container with the membrane on the rocker at room temperature for two hours.

After two hours, wash the membrane five times in 20 milliliters of PBST for five minutes each. Finally, add enhanced chemiluminescence substrate on the membrane. Then, expose the membrane to X-ray film according to the manufacturer's instructions.

This study is done to demonstrate the presence of SDS-resistant amyloid-like fibers in whole cell lysates obtained from tumor necrosis factor alpha-treated cells. It shows here that RIPK1 and RIPK3 show similar identical amyloid-like patterns. Interestingly, the MLKL fibers seem heterogenous with a different migration pattern than the others.

This image shows the possible migration pattern of amyloid or amyloid-like fibers. In first dimension SDD-AGE, amyloid or amyloid-like fibers exhibit a characteristic smear. In this image, the fibers migrate identically in the second run and exhibit a diagonal pattern of migration on the membrane at 45 degrees.

This shows that the fibers do not undergo degradation or dissociation during the gel running process. On the contrary, if the fibers do dissociate, there is vertical streaking below the diagonal line, indicating faster migration of the smaller fibers during second electrophoresis. Both these first and second dimension SDD-AGE show that the MLKL fibers do not dissociate during the SDD-AGE process and are indeed distinct from the RIPK1 and RIPK3 fibers.

In this image, the MLKL fibers show a characteristic smear during the first dimension. However, the same fibers show a sharp diagonal line with no vertical streaking in second dimension SDD-AGE. When attempting this procedure, it's important to remember that all conditions, such as voltage and length of the run, remain the same between the first and second dimensions to ensure a sharp line at a 45 degree angle.

Following this procedure, other methods, such as stripping the membrane and re-probing with other antibodies, can be performed to answer additional questions, such as if the fiber contains other proteins. After watching this video, you should have a good understanding of how to confirm that no degradation or dissociation of amyloid or amyloid-like fibers is taking place during traditional SDD-AGE.