The overall goal of this experiment is to determine the number of subunits in homooligomeric protein complexes in cell lysates using non-denaturing sodium dodecosulfate-polyacrylamide gel electrophoresis. This method can help answer key questions in the field of cell biology, such as the oligomeric status of proteins involved in fundamental cellular processes. The main advantage of this technique is that it does not require purification of the homooligomeric protein complexes or specialized, expensive equipment.
Therefore, it can be performed in most laboratories. This experiment utilizes two types of cells. A549 cells that express endogenous Myxovirus resistance A protein, or MxA;and virocells that overexpress MxA.
Seed the A549 cells and virocells into six well dishes with two milliliters of growth medium per well. Incubate the cells overnight in a cell culture incubator. On the following day, harvest the cells.
Wash each well with one milliliter of PBS and attach the cells by adding 0.5 milliliters of 0.25%Trypsin-EDTA solution for approximately five minutes at room temperature. As soon as the cells detach from the dish, add 0.5 milliliters of growth medium, and carefully mix by pipetting up and down. Transfer the cells of each well into a two milliliter tube, and pellet them using a table top centrifuge.
Carefully remove the supernatant by pipetting without disturbing the cell pellet. Wash the cells by adding one milliliter of ice cold PBS and carefully pipetting the cell suspension up and down. Pellet the cells in a table top centrifuge.
Carefully remove the supernatant by pipetting without detaching the cell pellet. Add 200 microliters of ice cold lysis buffer, resuspend the cells by pipetting up and down, and then place on ice. Immediately protect the lysate from light by covering the tubes with tinfoil because the iodoacetamide in the lysis buffer is light-sensitive.
Incubate on ice for 30 minutes. The use of iodoacetamide is crucial because iodoacetamide irreversibly protects the thiol group of free cysteines by forming a thioether bond and prevents artificial protein aggregation. Next, remove cell debris by centrifugation in a pre-chilled table top centrifuge.
During the centrifugation, prepare dialysis columns with a molecular weight cutoff of 10, 000. Attach the dialysis columns to a float buoy and place them into a beaker filled with pre-chilled dialysis buffer to equilibrate the columns. Do not touch the membrane.
Place a magnetic stirrer in a beaker and transfer the beaker into the cold room. Using the magnetic stirrer to ensure gentle stirring, equilibrate for 20 minutes at four degrees Celsius. After 20 minutes, remove the columns from the dialysis buffer and the float buoy.
Transfer the cleared lysates into the prepared dialysis columns by pipetting without touching the membrane. Attach the columns to a float buoy. Put them back into the beaker filled with dialysis buffer, and then transfer the beaker to the cold room.
Dialyze the lysate for at least four hours at four degrees Celsius while carefully stirring with a magnetic stirrer to remove small metabolites and salts that could interfere with non-denaturing polyacrylamide gel-electrophoresis. When the dialysis is complete, transfer each dialyzed sample to a 1.5 milliliter tube. Remove precipitates by centrifugation in a table top centrifuge.
Electrophoresis must be performed immediately after dialysis of the lysates to prevent the dissociation of the oligomeric protein complexes. Fill the inner and outer gel chamber with pre-chilled running buffer. Pre-run the non-denaturing polyacrylamide gel with pre-chilled running buffer at 25 milliamps per gel for 15 minutes at four degrees Celsius.
Mix 15 microliters of the prepared lysate with five microliters of 4X sample buffer. Load a native protein standard of choice and 15 microliters of sample on the gel. Run the gel at 25 milliamps for four hours at four degrees Celsius.
When the non-denaturing polyacrylamide gel electrophoresis is complete, disassemble the gel and carefully transfer into SDS buffer. Incubate for 10 minutes at room temperature with gentle shaking. Prepare two sponges, four cellulose filter paper sheets, and a blotting membrane per gel and soak them in blotting buffer.
Assemble the sandwich as follows from bottom to top. One sponge, two cellulose filter paper sheets, a blotting membrane, the gel, two cellulose filter paper sheets, and one sponge. Put the sandwich into the blotting tank, making sure the membrane faces the plus pole, while the gel faces the minus pole.
Fill the blotting tank with pre-chilled blotting buffer. For the best protein transfer results, blot at 90 milliamps overnight at four degrees Celsius. On the following day, disassemble the sandwich.
Incubate the membrane in Ponceau S solution for five minutes at room temperature to visualize the protein standard. Destain the membrane by placing it in deionized water and washing it. After transferring the membrane to a tray, mark the bands of the protein standard using a pen.
Block the membrane with blocking buffer for at least one hour at room temperature or overnight at four degrees Celsius. Subsequently, the proteins of interest are visualized by immunostaining using a rabbit polyclonal antibody directed against MxA as described in the text protocol. Virocells lacking endogenous MxA were transfected with recombinant wild type MxA and monomeric and dimeric MxA variance.
The migration of the recombinant MxA variance at their expected molecular weight confirmed their oligomeric state. The ectopically expressed wild type MxA migrated as a tetramer. These recombinant proteins were then used to assess the oligomeric state of endogenous human MxA protein derived from interferon-alpha stimulated A549 cells.
The result revealed that the size of MxA in lysates of interferon-alpha stimulated A549 cells corresponds to a tetramer. Once mastered, this technique can be done in one and a half days if it is performed properly. While attempting this procedure, it's important to remember not to freeze and thaw the samples during the entire procedure, since this could destroy protein interactions or cause protein aggregations.
Additionally, when working with light-sensitive reagents, like iodoacetamide, it is important to protect the samples from light during the lysis step. Following the non-denaturing SDS-PAGE, other methods like protein gel extraction and enzymatic activity AsiS or a second dimension PAGE can be performed in order to answer additional questions like enzymatic activity and the characterization of potential hetero-oligomeric complexes. After its development, this technique paved the way for researchers in the field of cell biology to explore the composition of oligomeric protein complexes in cultured cells.
After watching this video, you should have a good understanding of how to prepare cell lysates from cell cultures to study oligomeric proteins in non-denaturing PAGE analysis. Don't forget that working with iodoacetamide can be extremely hazardous and precautions such as wearing gloves and eye protection should always be taken.
