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14.2: Casting SDS-PAGE gels

  • Page ID
    17581
  • These instructions are designed for constructing two 12% SDS-PAGE gels with the BioRad Mini Protean system. One gel will be used for Simply Blue staining in the next lab. The second gel will be used for western blotting.

    Assemble the gel casting apparatus

    1. Assemble the components that you will need for casting the gel: a tall glass plate with attached 1 mm spacers, a small glass plate, a green casting frame and a casting stand.

    2. Place the green casting frame on the bench with the green “feet”resting firmly against the bench and the clamps open (perpendicular to the frame) and facing you.

    3. Place the two gel plates in the frame. Insert the taller spacer plate with the “UP” arrows up and the spacers facing toward you into the casting frame (the BioRad logo should be facing you). Insert the short glass plate in the front of the casting frame. There should be a space between the plates.

    4. Secure the plates in the casting frame by pushing the two gates of the frame out to the sides. IMPORTANT: the bottom edges of the two plates should be flush with the lab bench before you clamp the frame closed to ensure a watertight seal. To do this, rest the frame vertically on the bench BEFORE closing the gates.

    5. Clamp the casting frame with glass plates into the casting stand, with the gates of the casting frame facing you. Repeat steps 1-5 to prepare a second gel in the casting frame.

    6. Check to see if the assembled plates in the casting stand are sealed properly by pipetting a small amount of deionized water into the gap between the plates. If the glass plates hold water and don’t leak, you are ready to make the gels. Pour the water out by holding the entire cast- ing platform over a liquid waste container or sink. Use paper towels or tissues to absorb any residual water. If the gel leaks, disassemble the frame, dry the plates and go back to step 3.

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    Prepare two resolving gels.

    SAFETY NOTE:

    Acrylamide and bisacrylamide monomers are weak neurotoxins. Gloves and goggles should be used when working with acrylamide.

    ​​​​​​Assemble the chemicals that you will need to pour the gels. The table below shows the quantities of each chemical that you will need to pour two gels with the Mini-Protean system. Polymerization occurs rapidly, so be sure to follow the step-by-step instructions below.

    Note

    Catalysts should NOT be included into the mixture until you are ready to pour the gels!!

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    1. Label two 15 mL conical tubes “Resolving gel” and “Stacking gel”.

    2. Prepare ONLY the resolving gels at this time. Mix the acrylamide solution, pH 8.8 Tris buffer and water, as shown in the chart above. Mix the ingredients gently, trying not to introduce air. Oxygen inhibits polymerization of acrylamide gels.

    3. To the resolving gel mixture, add 100 μL of a 10% ammonium persulfate (APS) solution. Gen- tly mix the solution, trying not to introduce air. Oxygen inhibits acrylamide polymerization.

    4. Add 10 μL of TEMED catalyst. Once again, gently mix in the catalyst trying not to introduce air bubbles. CAUTION: TEMED has an unpleasant odor. Cover the tube immediately after you aliquot this reagent.

    5. Working quickly, use a plastic transfer pipette to fill the space between the two plates until the resolving gel solution reaches a height just above the green clamps on the gel casting frame. Draw up any remaining acrylamide into the transfer pipet. (You will know that the acrylamide has polymerized when you can no longer push the acrylamide out of the pipet.)

    6. Using a transfer pipet, add deionized water so that it gently flows across the surface of the polyacrylamide mixture. The water layer ensures that the polyacrylamide gel will have a level surface once it polymerizes.

    7. Allow the gel to polymerize, which takes ~15-20 minutes. You will note that the interface between the polyacrylamide and water overlay disappears temporarily while the gel polymer- izes. A sharp new interface then forms between the two layers, indicating that polymerization is complete. (You can also check the remaining polyacrylamide in the transfer pipette to see if it has polymerized.)

    8. When polymerization is complete, remove the water from the top of the resolving gel by tilting the gel to the side and using a paper towel or Kimwipe to wick out the water.

    Screen Shot 2019-01-04 at 8.09.08 PM.png

    Pour the stacking gels

    SAFETY NOTE: Be sure you are wearing goggles when pouring the stacking gels.

    1. Prepare the stacking gels. Mix the acrylamide solution, pH 6.8 Tris buffer and water, as shown in the chart above.

    2. Add 30 μL 10% APS and 7.5 μL TEMED to the stacking gel acrylamide mixture. Mix the contents by gently inverting the tube twice.

    3. Use a transfer pipette to pipette the stacking gel on top of the resolving gel between the two glass plates. Add enough stacking solution until it just reaches the top of the small plate.

    4. Carefully, but quickly, lower the comb into position, being careful not to introduce air bubbles. (The Bio Rad logo on the comb should be facing you.) Adding the comb will force some solution out of the gel, but this is fine. If air bubbles become trapped below the comb, remove the comb and reposition it.

    Save the SDS-PAGE gels

    1. Carefully remove the gels from the casting stand and then from their green frames.

    2. Keeping the combs in the gel, wrap the gels in a wet paper towel. Then wrap the gels in plastic wrap to be used in later labs. The gels will be ruined if they are not kept wet and properly wrapped!