The overall goal of this protocol is to enable researchers to capture, separate, and analyze native protein complexes extracted from tissue lysates using polyacrylamide gel electrophoresis. This method can aid researchers who study proteins because it is able to stabilize otherwise labile protein associations under near-native conditions, allowing for their identification and subsequent analysis. The main advantage of this technique is that it uses common biological methods so it is widely applicable and can be tailored to the goals of the individual researcher.
To begin this procedure, prepare for blue native polyacrylamide gel electrophoresis as described in the text protocol. Connect the electrodes to the power supply and electrophorese the proteins in the gel at 150 volts until the dye band progresses approximately two centimeters into the resolving layer. At this point, stop and disconnect the power supply.
After disassembling the electrophoresis apparatus, separate the glass panes of the gel cassette. Remove and discard the stacking layer. Carefully cut the gel just below the bottom edge of the dye front, taking care to make this cut as smooth and straight as possible.
Then, discard the unused piece of gel. Trim away any unused portions along the edges of the gel strip. Carefully place the strip into 10 milliliters of phosophate buffered saline in a small container and gently mix by notation for 30 minutes to equilibrate.
After equilibration, discard and replace the PBS with another 10 milliliters. Pipette 500 microliters of 25 millimolar DSP dissolved in dimethyl sulfoxide into the PBS and continue mixing as before for 30 minutes. After 30 minutes, pour off the DSP solution.
At 10 milliliters of 0.375 molar HSCL, pH 8.8, containing 2%sodium dodecyl sulfate to quench the unreacted DSP. Continue the notation for 15 minutes. While the gel strip is quenching, prepare SDS-PAGE gel solutions according to standard methods.
Do not add the polymerization reagents. After quenching, return the blue native gel strip to room temperature and cast the strip into a new gel cassette. To do this, carefully pick up the gel and place it onto a clean gel cassette spacer plate.
Orient the strip so that it is flipped from its prior orientation and the bottom of the dye front is nearest to the top of the new cassette. Place the strip such that its top edge lies even with where the top edge of the cover plate will be. Make sure the dye front is parallel to the horizontal edges of the glass plate.
Push one side of the excised strip against one of the spacer walls, leaving room on the other side for the gel to be poured and a protein standard or ladder to be loaded. If the bottom edge of the gel strip contains any jagged or uneven areas, carefully cut them away. Once the gel strip is positioned correctly, lay the cover plate over the spacer plate.
Apply gentle pressure to push out any trapped air bubbles. Continue to assemble the gel-pouring apparatus according to the manufacturer's instructions. The gel strip sometimes shifts when the top plate is being placed.
It helps to let the strip hang over the edge of the top plate a little so it can be adjusted with a spatula to correct any shifts. Add polymerization reagents to the resolving gel buffer and pour it into the prepared gel cassette using a serological pipette. Fill the gel cassette to approximately two centimeters below the excised blue native PAGE gel strip to leave room for the stacking layer.
Then, add 100 microliters of butanol over the top of the poured gel and allow 30 minutes for polymerization of the resolving layer before pouring off the butanol. Next, add the polymerization reagents to the stacking gel solution. Using a serological pipette, pour the stacking layer to fill all remaining empty space in the gel cassette.
Tilt the gel cassette as the stacking layer is poured so it fills the space below the gel strip and air bubbles are not trapped. As the stacking gel buffer fills the empty space below the gel strip, gradually return the gel cassette to level footing. Continue to fill the empty space next to the excised gel strip with the stacking gel buffer until it nearly overflows.
Then, allow the stacking layer to polymerize for 30 minutes. After the stacking layer has polymerized, remove the gel cassette from the pouring apparatus, rinse it with distilled water, and assemble the electrophoresis apparatus according to the manufacturer's instructions. Fill the inner chamber completely with 1x SDS-PAGE running buffer.
Then, fill the outer chamber to the level indicated by the manufacturer. Load the space next to the excised gel strip with a molecular weight ladder or the appropriate protein standard. Attach the electrodes to the power supply and electrophorese the samples at 120 volts.
When the Coomassie dye runs off the gel, stop the run and disconnect the power supply. Analyze the gel using standard methods of electroblotting and antibody-based protein detection. Validation of multimer-PAGE is shown here via immunoblot.
The captured kinesin complex is cleaved by diphyo-3-itol showing that the high molecular weight aggregates are formed by cross-linking smaller substituents. Depicted here, brain lysates treated with increasing concentrations of SDS or Triton increased detection of monomeric alpha-synuclein while decreasing the detection of the soluble oligomer tetromer. Here, representative results of multimer-PAGE used to capture soluble complexes in rat brain lysate are shown.
Proteins were detected via immunoblot. The expected monomeric weight of each protein is shown below the lanes. Similarly, capture of membrane-bound complexes is shown here.
Multimer-PAGE occasionally fails to capture a complex, as is shown on the SDHA respiratory complex two blot. A discussion of possible explanations for this can be found in the text protocol. Once mastered, this technique can be done in about eight hours if it is performed properly.
After watching this video, you should have a good understanding of how to perform in-gel cross-linking of native protein complexes followed by gel re-casting and separation by secondary SDS-PAGE. Don't forget that working with acrylamide can be extremely hazardous and personal protective equipment should be worn while casting the gel. Following this procedure, the separated stabilized complexes can be eluted or cut from the gel and can be analyzed by many means, including immunodetection or mass spectrometry depending on the researcher's needs.
