How to Make Polyacrylamide Gels


Although you can use polyacrylamide gels for separation of DNA, they are better suited for protein separation and characterization. They are widely used to study molecular weight, charge, structure and purity of proteins. The electrophoretic mobility of proteins enables them travel through the network of acrylamide polymers when current is applied. Proteins separate according to their size.

Things You'll Need

  • Glass plates
  • Comb
  • Spacer
  • Clamps
  • Syringe
  • Safety gloves
  • 150 mL flask
  • 26.6 mL of 29% Acrylamide plus 1% N,N-Methylenebisacrylamide
  • 52.7 mL of water
  • 20 mL of 5X Tris/BorateEDTA (TBE) Buffer
  • 0.7 mL of 10% Ammonium Persulfate
  • 35 micro liters of TEMED
  • Put on gloves. Thoroughly clean and dry the glass plates, comb and spacers.

  • Put the larger plate on a clean dry surface. Place spacers on both sides and cover with smaller plate. Carefully align the plates with the spacers on two sides and the bottom. Clamp the three sides of the plates to seal and hold them together.

  • Calculate the amount of gel volume required for the size of plates and thickness of spacers.

  • Determine percent of polyacrylamide solution needed for your experiment. For 100 mL of a 8% polyacrylamide gel, mix 26.6 mL of 29% Acrylamide plus 1% N,N-Methylenebisacrylamide, 52.7 mL of water, 20 mL of 5X TBE and 0.7 mL of 10% Ammonium Persulfate in a small flask. Add 35 micro liters of TEMED to the solution and mix gently. Work quickly after adding TEMED. Fill a syringe with the solution and carefully dispense contents in empty space between plates until solution is slightly below smaller plate.

  • Carefully insert the well-forming comb between the large and small plates on the top of the assembled plates. Let the gel polymerize completely for about an hour.

  • Gently remove the comb. Flush the wells with 1X TBE to clean them. Take off the clamps and place the gel in the electrophoresis apparatus according to manufacturer's directions. The polyacrylamide gel is ready to use for your experiment.

Tips & Warnings

  • Clean the glass plates thoroughly before pouring the gel. Any smudges or oil on the plated will result in air bubbles and hinder sample migration.
  • Because acrylamide is toxic, take upmost care when you work with it. Always wear gloves when handling acrylamide in any form. Use an automatic pipette to measure acrylamide solution and put on a face mask when using acrylamide powder.

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