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now: Sun Apr 11 00:24:19 2021 ... mod: Sun Apr 4 18:41:33 2021
Multiphasic Buffer Systems
Multiphasic buffer systems are electrophoretic techniques that utilize non-equilibrium buffer zones to enhance resolution. The gel is divided into two parts
- the stacking gel is a low-density, non-separating gel used to condense protein samples into a sharp band
- the resolving gel is a higher density gel used to separate the condensed proteins accoring to size, shape and charge
In the stacking gel, the migrating ions of the electrode buffer solution have different charge densities forming a leading front of highly charged ions, followed by a trailing front of less highly charged ions. The separation of these two fronts creates a large voltage gradient in which the protein sample is trapped, compressing the sample into a narrow band. When the trailing ionic front encounters the different conditions in the resolving gel, the trailing ions increase their charge density and accelerate, rapidly passing by the protein sample. No longer trapped between the two ionic fronts, the proteins are then free to separate from each other in the resolving gel. For example, in the first recipe below, the leading ion is chloride (Cl-) and the trailing ion is glycine. At pH 8.3 glycine carries a net charge of -0.048, while at pH 9.5 glycine has a net charge of -0.44.
These systems consist of recipes for stacking and resolving acrylamide gels and electrode buffer solutions at different pH's appropriate for resolution of native proteins.
make each solution up to 1 liter total volume with H2O
for proteins with pI below that of the stacking buffer: run to (+):
- stack pH 8.3, resolve pH 9.5
- stack: 32 ml 1M H3PO4, 7.13 g Tris
- resolve: 60 ml 1M HCl, 45.75 g Tris
- electrode buffer: 2.88 g glycine, 0.6 g Tris
- stack pH 7.0, resolve pH 8.0
- stack: 48.75 ml 1M H3PO4, 6.19 g Tris
- resolve: 60 ml 1M HCl, 8.56 g Tris
- electrode buffer: 5.52 g diethyl-barbituric acid, 1.0 g Tris
Initiate polymerization with ammonium persulfate and TEMED.
for proteins with pI above that of the stacking buffer : run to (-)
- stack pH 5.0, resolve pH 3.8
- stack: 60 ml 1M KOH, 3.59 ml CH3COOH
- resolve: 60 ml 1M KOH, 21.5 ml CH3COOH
- electrode buffer: 3.12 g β-alanine, 0.8 ml CH3COOH
- stack pH 4.0, resolve pH 2.3
- stack: 60 ml 1M KOH, 3.7 ml CH3COOH
- resolve: 15 ml 1M KOH, 66.6 ml CH3COOH
- electrode buffer: 2.8 g glycine, 0.3 ml CH3COOH
Initiate polymerization with 0.0125% ascorbic acid, 0.0003125% FeSO4
, 0.0015% H2
- The well-known Laemmli system for SDS-PAGE is an example of a multiphasic buffer system.
- Since this is a non-equilibrium method, never pre-run the gel. Doing so will destroy the stacking effect.
- Proteins will not migrate faster than the ionic front. Since tracking dye typically runs with the ionic front there is nothing to be gained by stopping the gel before the tracking dye has run off.
- use bromophenol blue as a negatively charged tracking dye
- use methyl green as a positively charged tracking dye (0.002% final concentration)