Team:Groningen/Normal SDS-PAGE using Tris-Glycine Gels and Electrophoresis Buffer


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Wear gloves at all times!


  • 30% acrylamide(Biorad) NEUROTOXIN!
  • 4X separation gel buffer: 1.5M Tris.HCl, 0.4% SDS pH6.8
  • 4X stacking gel buffer: 0.5M Tris.HCl, 1.92M glycine, 1% SDS water saturated isobutanol
  • TEMED( in yellow cabinet )
  • 10% APS (Ammonium persulfate) ; prepare 10 ml and keep cold in fridge

Hoeffer Mighty gel system Casting of the gel:

  • Per gel take one small glass plate with 'ears', one big white ceramic plate, two grey spacers with perpendicular plastic bits (0.75 mm), and one white comb (0.75 mm)
  • Clean the plates etc with soap, rinse with demiwater and ethanol, and dry
  • Assemble the system in the gel casting holder. Mark the line of separation/stacking
  • Mix the separation gel in 10 ml plastic tube:
                              for 2 gels
Seperation gel (0.75 mm)               12.5%            16%    
   30% acrylamide                      4 ml             5.1 ml
   4X separation buffer                2.4 ml           2.4 ml
   MQ                                  3.2 ml           2.1 ml
   10% APS                             28 μl            28 μl
   TEMED                               28 μl            28 μl
  • Pipet the separation gel mix immediately in between the glass plates until the marked line is reached
  • Pipet water-saturated isobutanol on top of the polymerizing separation gel
  • Let the separation gel polymerize completely before preparing the stacking gel
  • When the gel is polymerized, discard the isobutanol and wash the gel with water
  • Mix the stacking gel in a 10 ml tube:
                               for 2 gels
   mQ                               1.92 ml
   4X stacking buffer               0.83 ml
   30% acrylamide                   560 μl
   10% APS                          14 μl
   TEMED                            7 μl
  • Pipet the stacking gel mix immediately on top of the separation gel and place the comb without air bubbles. Mark the teeth of the comb with a marker
  • Let the gel polymerize (5-30 min); check also the remaining gelmix in the tube
  • Disassemble the gel casting holder and take out the gel/plates
  • Use the gel immediately or seal the gel in plastic seal bags and store at 4 °C

Assembly of the Tris-Glycine gel in the electrophoresis unit

  • Take the electrophoresis unit of the Hoeffer system and place it next to a power unit
  • Dilute 10X and pour 1X electrophoresis buffer in the container( roughly 1 cm high)
  • Place gel in the buffer without air bubbles under the gel
  • Use the red clamps to place the gel tightly in the unit
  • Pour 1X electrophoresis buffer in the chamber so that the top of the gel is immersed
  • Take the comb out of the gel and rinse the wells using a hooked needle and syringe

Runninge of the gel

  • Prepare the samples in 1X SDS sample buffer (NOT nucleic acid loading buffer)
  • Boil the samples for 5 min and spin down
  • Pipet the samples and protein marker carefully into the wells
  • Place the electrode cap on the unit and press lightly.Put the other side of the electrode cables in the correct holes of the power unit
  • Switch on the power unit and run until the blue front is appr. 1 cm from the end of the gel. Alternatively, use the prestained protein marker to identify the exact point of stopping
  • Disassemble the electrophoresis unit and take the gel
  • Take one plate off and cut one corner away for positioning purpose
  • Carefully bring the gel into a clean staining container using some demi water and/or spacers
  • Stain the gel with CBB or silver