Electrophoresis
Gel Preparation for SDS-PAGE
Two categories of buffer systems are available for SDS PAGE: continuous and discontinuous. Continuous systems use the same buffer in both the gel and tank. While continuous gels are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels. Discontinuous buffer systems employ different buffers for tank and gel, and often two different buffers within the gel, with a third buffer in the tank. Discontinuous systems concentrate, or "stack" the protein samples into a very narrow zone prior to separation, which results in improved band sharpness and resolution.
In the classic SDS PAGE system developed by Laemmli, the gel is divided into an upper "stacking" gel of low percentage (i.e. large pore size) and low pH (6.8) and a resolving gel with a pH of 8.8 with a much smaller pores. Both gels contain only Cl- as the mobile anion. The tank buffer has glycine as its anion, at a pH of 8.8. When electrophoresis begins, glycine enters the stacking gel, where equilibrium favors the zwitterionic form with zero net charges. The glycine front moves slowly through the stacking gel, lagging behind the strongly charged, smaller Cl- ions. As these two current-carrying species separate, a region of low conductivity, with a consequent high voltage drop, is created between them. This zone (a Kohlrausch discontinuity) "sweeps" the proteins rapidly through the large pores of the stacking gel, collecting the sample and depositing it at the top of the resolving gel in a focused narrow band. When the Kohlrasch discontinuity enters the resolving gel, the increase in pH ionizes the glycine so that it runs faster, dissipating the discontinuity. This allows the proteins to unstack and separate through the small pore resolving gel.
Casting a Discontinuous SDS-PAGE Gel (The Laemmli System)
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- Prepare to resolve gel and stacking gel casting solutions
The table below gives the formulations for SDS-PAGE resolving gels from 6 - 16% as well as the formulation for the stacking gel using National Diagnostics' ProtoGel family of products (or powdered reagents).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. To de-gas, stir the solution under aspiration for 10 minutes at room temperature.
- Prepare to resolve gel and stacking gel casting solutions
Resolving Gel Formulation
| % Gel | Size Range (kd) | Method 1: Volume of ProtoGel, ProtoGel Buffer to Use | Method 2: Volume of ProtoGel and Reagents to Use |
|---|---|---|---|
| 6 | 60-200 | Protogel: 20.0 mL ProtoGel Buffer: 25.0 mL Deionized H2O: 53.9 mL |
ProtoGel: 20.0 mL 1.5 M Tris-HCl (pH 8.8): 25.0 mL 10% SDS: 1.0 mL Deionized H2O: 52.9 mL |
| 8 | 40-140 | Protogel: 26.7 mL ProtoGel Buffer: 25.0 mL Deionized H2O: 47.2 mL |
ProtoGel: 26.7 mL 1.5 M Tris-HCl (pH 8.8): 25.0 mL 10% SDS: 1.0 mL Deionized H2O: 46.2 mL |
| 10 | 20-80 | Protogel: 33.3 mL ProtoGel Buffer: 25.0 mL Deionized H2O: 40.6 mL |
ProtoGel: 26.7 mL 1.5 M Tris-HCl (pH 8.8): 25.0 mL 10% SDS: 1.0 mL Deionized H2O: 46.2 mL |
| 12 | 15-70 | Protogel: 40.0 mL ProtoGel Buffer: 25.0 mL Deionized H2O: 33.9 mL |
ProtoGel: 40.0 mL 1.5 M Tris-HCl (pH 8.8): 25.0 mL 10% SDS: 1.0 mL Deionized H2O: 32.9 mL |
| 15 | 15-50 | Protogel: 50.0 mL ProtoGel Buffer: 25.0 mL Deionized H2O: 23.9 mL |
ProtoGel: 50.0 mL 1.5 M Tris-HCl (pH 8.8): 25.0 mL 10% SDS: 1.0 mL/ Deionized H2O: 22.9 mL |
| Stacking Gel Formulation | |||
| Protogel: 2.6 mL ProtoGel Stacking Buffer: 5.0 mL Deionized H2O: 12.2 mL |
ProtoGel: 2.6 mL 0.5 M Tris-HCl (pH 8.8): 5.0 mL 10% SDS: 1.0 mL Deionized H2O: 11.6 mL |
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- Pour the resolving gel: Add 1.0ml of fresh 10% ammonium persulfate solution for every 100ml of the casting solution. Swirl gently to mix. Add 0.1ml of TEMED for every 100ml of the casting solution. Swirl gently to mix. Pour the solution into the gel cassette. Fill the cassette to a level that 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.
- Pour the stacking gel: Rinse the butanol from the top of the gel with water, and drain the water by inverting the gel. Add 0.2 ml of 10% ammonium persulfate and 20 µl TEMED for every 20 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 SDS.
Casting Tris-Tricine Gels
Discontinuous SDS-PAGE employing Tris-Glycine-SDS as the tank buffer, the Laemmli system, resolves proteins down to about 15 kd. However, below this size, the proteins do not "destack" from the SDS micelles running through the gel with the buffer front. In order to resolve proteins in this size range, the Tris-Tricine system of Schagger and von Jagow (1987) was developed.
An Alternative to the Schagger and von Jagow System Running Tris-Tricine Gels using National Diagnostics 10X Tris-Tricine-SDS Buffer
With National Diagnostics 10X Tris-Tricine-SDS Buffer, you can extend the range of SDS-PAGE to resolve smaller proteins with minimal alteration of the protocol. To provide this level of convenience, National Diagnostics streamlined the original method of Shaggar and Von Jagow (1987 Anal. Biochem 166) by developing 10X Tris-Tricine-SDS cathode tank buffer to be compatible with the standard Laemmli gel/buffer system. This combination resolves proteins as small as 5kD. The researcher simply substitutes National Diagnostic's 10X Tris-Tricine-SDS Buffer in the upper (cathode) tank, with no other changes from the standard Laemmli protocol, to extend the resolution of their gels.
The Original Schagger and von Jagow System
Tricine gels of the Schagger and von Jagow System are poured in the same manner as Laemmli gels. However, unlike the Laemmli system, the stacking and resolving gels are poured using the same Laemmli buffer concentrate:
Buffer concentrate:
Resolving gel:
Stacking gel:
- 3ml buffer concentrate
- 1.6ml ProtoGel
- 7.5ml H2O
Polymerize in an ordinary manner and run with 0.2M Tris-HCl pH 8.9 in the lower chamber, and 0.1M tris, 0.1M tricine and 0.1% SDS in the upper chamber.
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