See also capillary electrophoresis under Diagnostic Techniques and Common Analytical Techniques

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells) to investigate subunit compositions and to purify the protein for subsequent applications. In polyacrylamide gel electrophoresis, proteins migrate due to an electrical field through pores in the gel matrix. Pore size decreases with higher acrylamide concentrations. The migrations of the protein is determined by the gel pore size and protein charges, size and shape.

Isolectric Focusing:

The basis principale behind isoelectric focusing in a pH gradeint is that a charged molecule will become immobilzied in an electric field when it migrates to a position in the pH gradient that is equal to its isoelectric point (zero net charge). This process occurs independently of the initial location of a specific prtoein in the solution. It is the result of the disappearance of the effective electrical charge of the protein when migrating to the region where pH is equal to pH. (Zilberstein, US 7914656)

One dimensional gel (1-D) electrophoresis

1-D electrophoresis under denaturing conditions (i.e., in the presence of 0.1% SDS) separates proteins based on molecular size. Most proteins bind SDS in a constant weight ratio, leading to identical charge densities for the denatured proteins. Thus the SDS protein complexes migrate in the polyacrylamide gel according to size, not charge. Most proteins are resolved on polyacrylamide gels containing from 5% to 15% acrylamide. The relationship between the relative mobility and log molecular weight is linear over these ranges. After the proteins are solubilized by boiling in the presence of SDS, an aliquot of the protein solution is applied to a gel lane, and the individual proteins are separated electrophoretically.  2-mercaptoethanol is added during solubilization to reduce disulfide bonds. Comparison of reducing and nonreducing gels can provide valuable information about the number of disulfide cross linked subunits in a protein complex. If the subunits are held together by disulfide linkages, the protein will separate in denaturing gels as smaller sized subunits as compared to nonreducing conditions where the protein will separate as a complex.

The polyacrylamide gel is cast as a separating gel topped by a stacking gel and secured in an electrophoresis apparatus. After leaving the stacking gel, the protein enters the separating gel which has a smaller pore size, a higher salt concentration and higher pH compared to the stacking gel. The proteins are separated according to either molecular size in a denaturing gel (containing SDS) or molecular shape, size, and charge in a nondenaturing gel.

Two-dimensional  (2-D) gel electrophoresis

2-D gel electrophoresis separates proteins in the first dimension by isoelectric focusing and in the second dimension by electrophoresis in the presence of SDS. Thus information is obtained not only about size as in one-dimensional gels but also about the charge of the protein. In isoelectric focusing a pH gradient is established using ampholines. Proteins migrate to their isoelectric point (pH) at which there is no net charge.

The protein spots can be visualized by staining. An imaging system is used to record an image of the stained gel to provide a record of the protein distribution in the sample. These images can be analyzed, compared, and archived with software packages (e.g., MELANIE, PDQuest, Z3, Progenesis Workstation, ProteomeWeaver, ProteinMine, Delta2D, DeCyder).

Challenges: A concern with 2D gels is that despite their resolving power, 2D gels do not completely resolve all proteins into single spots. Many spots contain 2-5 proteins. Another concern is that even with the most sensitive stains, there is a limited dynamic range for protein detection. Cellular expression levels of different proteins can differ by as much as a million-fold whereas the dynamic range for protein staining is about 100-2000 fold. Thus, 2D gels typically detect only the most abundantly expressed proteins.

2-D in Combination with other Techniques

2D-SDS-PAGE combined with a high-throuput MS analytical method (MALDI-TOF MS). Gel digestion is used to cleave the proteins to peptides which are then analyzed by MALDI-TOF MS. The MS data is then analyzed with a peptide mass fingerprinting algorithm and software which identifies the proteins present. A second approach is to use peptide sequence identification by tandem MS. Here, one digests the proteins in the mixture to peptides which are then resolved (at least partially) by chromatography and then electrospray tandem MS. These spectra are mapped to protein sequences from databases with the aid of Sequest or similar search tools. The 2 distingushing characteristics of this approach are 1) the analysis primarily involves working with peptides rather than with proteins and the protein identification is based on the MS-MS fragmentation spectra, rather than on peptide mass fingerprinting.

Fractionation of Proteins by pI value and then 2-D: Hagner-mcwhirter (WO 2007/058584) discloses a method of loading a sample containing a complex mixture of proteins onto media which can be chromatography matrix and eluting by centrigual force with at least three buffers having different pH in a stepwise manner to obtain at least three fractions separated accordingl to pI value and then subjecting each fraction to further separation by 2D electrophoresis.