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Electrophoresis

Electrophoresis is commonly used to separate proteins on the basis of size and/or charge. Typically, a sample containing target proteins is loaded into a porous matrix and voltage is applied. The proteins in the sample will migrate through the matrix at different velocities based on their varying size and charge.

At the end of the separation, the proteins can be detected as bands located at different vertical positions in the matrix. The matrix can be composed of a number of different materials, including paper, cellulose acetate, or gels made of polyacrylamide, agarose, or starch.

Polyacrylamide gels are the most commonly used matrices for the separation of proteins in a process called polyacrylamide gel electrophoresis (PAGE).

SDS-PAGE Gel Type and Pore Size Buffer Systems Equipment Molecular Weight Markers

Since a protein’s net charge will affect its mobility in an electric field, the detergent sodium dodecyl sulfate (SDS) is usually added to denatured protein samples and buffers. This confers a negative charge to proteins roughly in proportion to polypeptide length, ensuring that mobility depends primarily on molecular mass and not charge.

Native (non-denaturing) gel electrophoresis is run in the absence of SDS, and thus the mobility of proteins depends on both charge and hydrodynamic size.

The resolution of SDS-PAGE depends on protein size and gel porosity. Several acrylamide concentrations should therefore be tested in order to optimize migration conditions. Polyacrylamide gels may be cast either as homogenous and gradient gels.

Homogeneous gels have a uniform polyacrylamide concentration throughout the gel, while gradient gels comprise a linear progression of acrylamide concentrations for a wider separation range, but with less resolution. Gel densities are affected by both the acrylamide (%T) and crosslinker concentrations: the higher the %T, the smaller the pore size and the more proteins are impeded in their migration.

A typical PAGE setup uses two gels, where a stacking gel is cast above a resolving gel. The stacking gel is homogenous and contains relatively large pores (%T 4% to 5%). During electrophoresis, this phase allows the protein sample to concentrate into a narrow band before it enters the resolving gel, thereby increasing band resolution. The %T in the resolving gel can range from 5% to 20%.

Use the chart below to estimate optimal %T in the resolving gel required for the molecular weight of your target protein.

Target Size Range (Mr)

%T in Resolving Gel

36 000 to 205 000

5%

24 000 to 205 000

7.5%

14 000 to 205 000

10%

14 000 to 66 000

12.5%

14 000 to 45 000

15%


GE Healthcare offers reagents and accessories for gel casting, as well as a range of precast polyacrylamide gels such as ExcelGel, PhastGel, and DALT Gel.

Proteins are characterized by a charge determined by the pH of the surrounding medium, and in consequence, the ionization status of their carboxyl and amino groups. Post-translational modifications may also affect the overall charge of a protein. The pH of the medium must thus remain constant during electrophoresis.

Two types of buffer systems, continuous and discontinuous systems, are commonly used. A continuous system uses the same buffer for both the tanks and gel. In a discontinuous system, the two gel layers are made with different buffers, and the tank buffers differ from the gel buffers. Continuous systems are slightly easier to set up and tend to suffer from fewer problems related to sample precipitation and aggregation. Discontinuous systems, on the other hand, provide increased protein resolution and are more widely applied.

A wide range of equipment for running polyacrylamide gels is available and the choice of equipment is usually based on the nature of the protein target, resolution requirements, and personal preferences. Whether you prefer precast or handcast gels, or vertical or horizontal electrophoresis systems, we have a range of equipment that make electrophoresis convenient and reproducible.

Our Vertical Systems include: miniVE, SE250 and SE260 mini-vertical units, as well as SE2600 Ruby, SE660 Dual Cooled, and SE400 vertical units.

In addition to our vertical systems, we offer Horizontal Flatbed Systems including Amersham ECL Gel electrophoresis system, Multiphor II, and PhastSystem.

The horizontal Amersham ECL Gel electrophoresis system, with precast gels, offers a number of advantages over vertical units, such as reducing buffer consumption and eliminating the risk of buffer spills from the top to bottom buffer chamber. In addition, the heat generated during electrophoresis is evenly distributed over the entire gel surface, reducing the risk of “smiling” gels.

Molecular weight markers are composed of a mixture of proteins of known size and the distances migrated over the time course of a PAGE run provide a logarithmic scale by which to estimate the size of unknown proteins in samples. For most runs, it is advisable to reserve at least one lane for the inclusion of molecular weight markers. Markers and samples should be exposed to similar conditions prior to PAGE.

In addition to size estimation, molecular weight markers allow you to monitor the progress of your proteins throughout electrophoresis as well as to assess transfer efficiency. Prestained and unstained molecular weight markers from GE Healthcare allow you to estimate the molecular weight of proteins separated by PAGE, from Mr 2500 to 669 000 (2.5 kDa to 669 kDa).

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