SDS-PAGE

Materials
  1. MilliQ H2O
  2. 30% acrylamide/0.8% bisacrylamide solution (4oC, alumnium shield)
  3. Separating (Running) gel buffer (lower Tris buffer)
  4. Stacking gel buffer (Up tris buffer)
  5. 20% ammonium persulfate(AP, 4oC, make fresh if more than 3 months)
  6. TEMED (4oC)
  7. H2O-saturated isobutyl alchohol
  8. 5X sample buffer (-20oC)
  9. Electrophoresis buffer
  10. Protein molecular-weight-standard mixture
  11. Mini-PROTEAN 3 Electrophoresis System
  12. 50ml Felcon tubes
  13. Ice bucket
  14. 10mml sereological pipets
  15. 10ul, 200ul, 1000ul pipetter with tips
  16. Samples to be analysed
Procedure
  1. Preparation of separating and stacking gels

    Cast and asembly following the tour from the manufacturer.

    Put stock solutions on ice before casting gels. For 40 ml separating gel, in 50ml Felcon tubes add solutions according to table 1. The gel percentage is based on the molecular weight of the protein to be analysed. Refer table 2 for guidance.

    Table 1. Separating gel and stacking gel's composition
    Stock solution Separating gel
    (Type gel percentage,
    then click OK    )
    %     		Stacking gel
    H2O
    ml
    11.6 ml
    Tris Buffer
    10 ml
    5 ml
    Acrylamide/Bis
    ml
    3.4 ml
    20% AP
    200 ul
    50 ul
    TEMED
    20 ul
    40 ul

    Table 2. The relation between separating gel's percentages and protein molecular weight size ranges
    % separating gel
    6
    8
    10
    12
    15
    Size range (KD)
    		60-200 40-140 20-80 15-70 10-50

    Swirl gently to mix. If there are bubles, vacum or centrifuge at 3000 rpm for 2-5 min at 0-4oC. Pour the solution into the gel cassette. Fill the cassette to a level which will allow the comb to be inserted with 5mm between the bottom of the wells and the top of the separating gel. Overlay the gel with water saturated n-butanol. Allow the gel to polymerize for 30 minutes. A line will become visible at the top of the gel as it polymerizes.

    Rinse the butanol from the top of the gel with water, and drain the water by inverting the gel.

    Prepare the stacking gel solution and fill the top of the cassette. Insert the comb. Allow the stacking gel to polymerize for 30-60 minutes .

    Romove the comb carefully and rinse the wells with running buffer.
  2. Preparation of samples
    For 15-well, 1.5 mm gel, take 10-15 ug/well prtein equevilent sample into 1.5ml Eppendorf tube on ice, add H2O to the same volume for all samples. add 5X sample buffer so that the final concentration to 1X. Mix well. Heat on 95oC thermal block for 5 min. Cool to room temperature.
  3. Running the gel
    Load the sample from step 2 onto the gel with gel loading tips.
    Make sure that the running buffer in the electrode assembly is higher than the top of shorter glass plates and the buffer outside the electrode assembly higher than the bottom of the plates at least one centimeter. Cover the lid with appropriate electrodes connected and run the gel at 190V until the blue dye close to or just out of the gel (about one hour).

    Disassemble the gel unit and take the gel(s) for Coomasse Blue staining or for Western blot.
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