Recipe for SDS PAGE Gels

Gel Calculator
by Axel Masanek
last changed 20.11.2007
Separating Gel Stacking Gel
Volume ml Volume ml
Concentration Separating Gel % Concentration Stacking Gel %
Concentration of your acrylamide solution %/ %
total acrylamide/bisacrylamide ratio =
Cross linking value= %
Your recipe
Separating Gel Stacking Gel
urea g
8×separating gel buffer ml 2×stacking gel buffer ml
acrylamide solution ml acrylamide solution ml
water ml water ml
Degass for 1 min! Add APS and TEMED immediateley before casting!
APS µl APS µl
TEMED µl TEMED µl

Acrylamide is highly toxic! For your health and the quality of your samples always wear gloves!

Your workflow

  1. Separating gel

    1. Clean all glass plates and spacers with ethanol.
    2. Assemble the gel caster according to the manual of the manufacturer.
    3. Ensure that the casting stand is located horizontally.
    4. Prepare the gel solution in a filtering flask according to the values of the gel calculator and mix it thoroughly.
      Verify that the urea is completely dissolved!
    5. Degass the solution for 1 min.
    6. Add TEMED and APS according to the values of the gel calculator and mix the solution.
    7. Pour the acrylamide solution in the gel caster or between the glass plates up to the desired height.
    8. Overlay the cast gel with n-butanol.
    9. The separating gel takes about 1 hour to polymerise.
      Longer polymerisation times e.g. overnight reduce the amount of free acrylamides in the gel and therefore the risk of the formation of acrylamide adducts, which impair somehow the MS analysis of protein spots from the gel.
    10. Remove the butanol by rinsing with water.
      Separating gels can be stored at 4°C for up to two weeks. Avoid drying by wrapping with wet paper towels.

  2. Stacking gel

    1. Clean comb with ethanol.
    2. Dry the area above the separating gel completely with Whatman paper.
    3. Fix gel in an upright position using e.g. the casting stand or clips.
    4. Place the comb in the gel sandwich.
    5. Prepare the stacking gel solution in a filtering flask according to the values of the gel calculator and mix it.
    6. Degass the solution for 1 min.
    7. Add TEMED and APS according to the values of the gel calculator and mix the solution.
    8. Pipette the gel solution onto the separating gel.
    9. The stacking gel takes about 30 minutes for polymerisation.

Buffers and Solutions
8 x Separating gel buffer Store at 4°C
for 1 l
3 M Tris pH 8.8 363.48 g
Adjust pH 8.8 with HCl
2 x Stacking gel buffer Store at 4°C
for 500 ml
0.25 M Tris pH 6.8 15.14 g
Bromophenol Blue (Stock 2% in H2O) 300 µl
Adjust pH 6.8 with HCl
APS Aliquot and store at -20 °C
for 10 ml
10 % (w/v) Ammonium persulfate 1 g