2-D Electrophoresis, Facts and Basic Data

2-D Electrophoresis Separations of Proteins, Nucleic Acids, Unknowns

Nov 11, 2009

Two-Dimensional Gel Electrophoresis, LMMB NCIFCRF U.S.

2-D electrophoresis adds a second dimension to the analysis of protein and DNA and, in proteomics, it helps scientists analyze and understand proteins, and the genome.

Proteomics is the biochemical study of the the full array of proteins that are expressed by the genome of certain cells, tissues or organisms. Not all genes of the genome are turned on at all times. Proteomics seeks to determine and define all the possible protein products, modified and unmodified, that may be produced by the genome. Electrophoresis that is 2-dimensional is very special and useful to this end. Also known as 2-D electrophoresis, this procedure is invaluable for understanding and characterizing genomes and proteins. Electrophoresis is the electric field-induced movement of molecules through a gel such as polyacrylamide, starch or agarose.

2-D Electrophoresis , Basic Facts, Data and Essentials

2-D electrophoresis has the ability to separate and resolve proteins in a very clear and distinct manner.

The first dimension run separates proteins on the basis of isoelectric points, i.e. that pH at which all charges on the molecules are neutralized and balanced (see photo 1 below). Essentially, there is no net charge at the electric point. Proteins vary in their isolelectric points, and most proteins have very distinct isoelectric points. Isoelectric points can be achieved by: 1. immobilized pH gradient electrophoresis (IPGE); 2. isoelectric focusing (IEF); or, 3. non-equilibrium pH gradient electrophoresis (NEPHGE). Under controlled conditions of temperature and urea concentration, many proteins attain focusing points by IPGE (and somewhat by IEF) that correspond to the isoelectric points determined by evaluation of the proteins' amino acid compositions.
The second dimension separation of the proteins is achieved by the proteins' molecular masses and their migration rates in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). This migration is shown in photo 1 below. The molecular weights determined by SDS-PAGE are accurate in most cases(+/- 10%), with a few, occasional stark exceptions.

Two-Dimensional Electrophoresis Facts and Example Relating to Proteonomics

A 2-D gel electrophoresis and proteonomics research study by Hurkman, W.J. and Tanaka, C.K. 2004 is a very good example of excellence and application of these techniques in biology (Improved methods for separation of wheat endosperm proteins and analysis by two-dimensional gel electrophoresis. J. Cereal Sci. 40: 295-299). That research study is Illustrated by photos below (photos 2-5,below, click to enlarge), and a summary of several of the important steps in proteomics (protein analyses of the genome products in cells) is provided in sequence here:

laboratory extraction of the wheat endosperm proteins is done.
protein extract is added to the gel well, and 2-D gel electrophoresis is done (photo 1) .
separated proteins are stained with Coomassie Blue (photo 2).
computer software is used to analyze the densities and locations of the spots (photo 3).
robotics are used to: punch out of the spots, pick them up, and place them in wells of plastic plates (photo 4).
enzymatic spot digestion occurs, peptides are produced, and automated anlayses by mass spectroscopy is the final step (photo 5).

Two-Dimensional Electrophoresis Concluding and Summary Ideas

The genome is not operating all genes at once. In fact, in some cases, many genes may be turned off. The goal of genetic and biological studies is to determine all the genes, all the gene's products and, ultimately, to try to explain what the organism is and what it can do. Science is at the dawning of that era. Much has been uncovered, and much need to be.

Consider a career in genetics, biochemistry or biology, or one of the allied health sciences. The future is bright for scientific inquiry and scientific and medical applications in the sciences.

Sources

Lodish, H. et al. 2000. Molecular Cell Biology. Fourth Ed., W. H. Freeman and Co., New York, N.Y.

Wandersee, J.H., et al., eds. 1996. Bioinstrumentation, Tools for Understanding Life. National Assoc. Biol. Teachers, Reston, Va.

The copyright of the article 2-D Electrophoresis, Facts and Basic Data in Scientific Research Methods is owned by Donald Reinhardt. Permission to republish 2-D Electrophoresis, Facts and Basic Data in print or online must be granted by the author in writing.