Multimer-PAGE for Separating Native Protein Complexes: A Hybrid Separation Technique Consisting of Blue Native-PAGE and SDS-PAGE to Separate Intact Multimeric Proteins From Tissue Lysate
Transcripción
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 2 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 phosphate buffered saline in a small container, and gently mix by nutation 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. Add 10 milliliters of 0.375 molar Tris-HCl, pH 8.8, containing 2% sodium dodecyl sulfate, to quench the unreacted DSP. Continue the nutation 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 2 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 spaces 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.
