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Gel Electrophoresis of Proteins

Gel Preparation for Native Protein Electrophoresis

The basic protocols for preparing Native PAGE gels are the same as for discontinuous SDS PAGE gels, substituting non-SDS buffers for those containing SDS, as follows:

Casting Native Protein Gels

  1. Prepare resolving gel and stacking gel casting solutions
    The table below gives the formulations for native resolving gels from 6 - 12% as well as the formulation for the stacking gel.

    Formulations of Native Protein Gels

    Gel % ProtoGel (mL) Resolving Gel Buffer (mL) Deionized Water (mL)
    6 2 2.5 5.5
    8 2.7 2.5 4.8
    10 3.3 2.5 4.2
    12 4 2.5 3.5
    Stacking Gel 1.25 2.5
    (Stacking Gel Buffer)

    The 4X resolving buffer should consist of 1.5M Tris HCl with pH 8.8, the 4X stacking gel buffer should consist of 0.6M Tris HCl at pH 6.8 and the 10X running (tank) buffer should consist of 1.92M glycine and 0.25M Tris, at pH 8.3.

    Formulate enough resolving gel solution to fill the cassette and formulate 1/5 that amount of stacking gel solution. De-gas the solutions for optimum reproducibility. Stir the solution under aspiration for 10 minutes at room temperature.

  2. Pour the resolving gel
    Add 0.1ml of fresh 10% ammonium persulfate (APS) solution for every 10ml of casting solution. Swirl gently to mix. Add 10 µl of TEMED for every 10ml of casting solution. Swirl gently to mix. 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 resolving gel. Overlay the gel with 1-2mm of water saturated n-butanol to exclude O2 and ensure a flat interface between the resolving and stacking gels. Allow the gel to polymerize for 30 minutes. A line will become visible at the top of the gel as it polymerizes.
  3. Pour the stacking gelRinse the butanol from the top of the gel with water, and drain the water by inverting the gel. Add 0.1 ml of 10% APS and 10 µl TEMED for every 10 ml of stacking gel solution and fill the top of the cassette with this mixture. Insert the comb until the teeth are 5mm from the resolving gel. The comb should rest so that the tops of the well dividers are level with the top of the short plate. This excludes oxygen while ensuring that the dividers will fully separate the wells. Allow the stacking gel to polymerize for 30-60 minutes. Run the gel in 1X Tris-Glycine buffer (see above).

Native Gradient Gels

Native gradient gels use the same pouring apparatus and techniques as SDS PAGE gradient gels. The table below gives the formulations for the high and low percentage solutions needed to formulate native PAGE gradient gels.

Native Gradient Gel Component Solutions

Monomer % ProtoGel (30% 37:1 Acrylamide:MBA) (mL) Buffer (1.5M Tris-HCl pH 8.8) (mL) Deionized Water Sucrose (g)
Upper Solutions 4 2.6 5 12.4 ---
6 4 5 11 ---
8 5.3 5 9.7 ---
10 6.7 5 8.3 ---
Lower Solutions 10 6.7 5 6.7 3
12 8 5 5.3 3
15 10 5 3.3 3
20 13.3 5 0 3

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