 |
What you need to know about 2-D electrophoresis
Seven steps to successful results
1. Sample preparation
2. First dimension - IEF
3. Second dimension - SDS-PAGE
4. Gel staining
5. Detection
6. Image analysis
7. Spot processing for MS analysis
Introduction to two-dimensional (2-D) electrophoresis
Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples. This technique separates proteins according to two independent properties in two discrete steps:
- The first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI).
- The second-dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights (MW).
Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample. Thousands of different proteins can thus be separated, and information such as the pI, apparent mw, and the amount of each protein is obtained.
Two-dimensional electrophoresis was first introduced by O'Farrell and J. Klose in 1975. In the original technique, the first-dimension separation was performed in carrier ampholyte-containing polyacrylamide gels cast in narrow tubes. (Under the influence of an electric current, carrier ampholytes form a pH gradient, a critical component of IEF). Sample was applied to one end of each tube gel and separated at high voltages. After IEF the gel rods were removed from their tubes, equilibrated in SDS sample buffer, and placed on vertical SDS-polyacrylamide gels for the second-dimension separation.
The power of 2-D electrophoresis as a biochemical separation technique has been recognized since its introduction. Its application, however, has become increasingly significant in years as a result of a number of developments.
The 2-D technique has been improved to generate 2-D maps that are superior in terms of resolution and reproducibility. This new 2-D technique, developed by A. Görg and colleagues utilizes an improved first-dimension separation method that replaces the carrier ampholyte-generated pH gradients with immobilized pH gradients (IPG) and replaces the tube gels with gel strips supported by a plastic film backing.
Methods for the rapid analysis of proteins have been improved to the point that single spots eluted or transferred from single 2-D gels can be rapidly identified. Mass spectrometry techniques have been developed that allow analysis of very small quantities of peptides and proteins. Chemical microsequencing and amino acid analysis can be performed on increasingly smaller samples. Immunochemical identification is now possible with a wide assortment of available antibodies.
More-powerful, less expensive computers and software are now available, allowing routine computerized evaluations of the highly complex 2-D patterns.
Data about entire genomes (or substantial fractions thereof) for a number of organisms are now available, allowing rapid identification of the gene encoding a protein separated by 2-D electrophoresis.
The internet provides simple, direct access to spot pattern databases for the comparison of electrophoresis results and also to genome sequence databases for assignment of sequence information.
A large and growing application of 2-D electrophoresis is "proteome analysis", also called proteomics Proteome analysis is "the analysis of the PROTEin complement expressed by a genOME." The analysis involves the systematic separation, identification, and quantitation of many proteins simultaneously from a single sample. 2-D electrophoresis is used in this application due to its unparalleled ability to separate thousands of proteins. 2-D electrophoresis is also unique in its ability to detect post- and cotranslational modifications, which cannot be predicted from the genome sequence.
Other applications of 2-D electrophoresis include analysis of cell differentiation, detection of disease markers, monitoring therapies, drug discovery, cancer research, purity checks, and microscale protein purification.
See our product selection for each 2-D electrophoresis workflow step
Share results on our online 2-D discussion forum |
|
