Proteomics/Protein Separations - Electrophoresis/Introduction to Electrophoresis

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Introduction to Electrophoresis

Definition: e•lec•tro•pho•re•sis (ĭ-lĕk'trō-fə-rē'sĭs) n.

As given by American Heritage Dictionaries

1) The migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes. Also called cataphoresis.

2) A method of separating substances, especially proteins, and analyzing molecular structure based on the rate of movement of each component in a colloidal suspension while under the influence of an electric field.

Electrophoresis Theory

The separation by electrophoresis depends on differences in the migration velocity of ions or solutes through the given medium in the electric field. The electrophoretic migration velocity ( $u_{p}$ ) of an analyte is:

$u_{p}=\mu _{p}E$

Where E is the electric field strength and $\mu _{p}$ is the electrophoretic mobility.

The electrophoretic mobility is inversely proportional to frictional forces in the buffer and directly proportional to sample's the ionic charge. The forces of friction against an ion is dependent on size of the ion and the viscosity (η) of the medium. Analytes with different frictional forces or different charges will separate from one another when they move through a buffer. At a given pH, the electrophoretic mobility of an analyte is:

$\mu _{p}={\frac {z}{6\pi \eta r}}$

Where r is the radius of the analyte and z is the net charge of the analyte.

The differences in the ratio of charge to size of the analytes cause differences in electrophoretic mobility. Small and highly charged analytes have greater mobility, whereas large and low charged analytes have lower mobility.

Electrophoretic mobility is an indication of speed of a given analyte in a give medium. It is the balance of electrical force that acts in favor and the frictional force that acts against the motion. These two forces remain steady during electrophoresis; therefore electrophoresis mobility is a constant for a given ion under a given set of conditions. Based on this characteristic property of ion or solute, it can be separated using electrophoresis.

Applications of Electrophoresis

Electrophoresis has wide variety of applications in proteomics, forensics, molecular biology, genetics, biochemistry, and microbiology.

The common method of electrophoresis used in proteomics is Polyacrylamide Gel Electrophoresis (PAGE). PAGE is used for separation, identification, and purification of proteins. The proteins can be analyzed for information about the mass, charge, and purity. Different types of PAGE provide different types of information about the proteins. SDS-PAGE is used for separating proteins based on their molecular mass. SDS-PAGE is also used for protein identification and quantitation, identification of disulfide bonds,determination of purity of sample, and also blotting applications. Native-PAGE is used to separate proteins in their native form without denaturing. QPNC-PAGE is used to isolate active or native metalloproteins in biological samples. 2D-PAGE separates modified proteins and provide information about various modified states. 2D-PAGE and DIGE are used to study differential expression of proteins in healthy and diseased tissues. Proteins that are more abundant in disease tissue may be considered as diagnostic disease markers or potential drug targets. Since a sinle 2-DE can resolve thousands of proteins it is also used for cell map proteomics as well as for characterizing subproteomes. Capillary electrophoresis is employed for faster, efficient separation of proteins.

Electrophoresis is also used for separation and analysis of DNA. Sequencing of DNA, analysis of PCR products, separation and estimation of the size of the DNA fragments from restriction enzyme digestion are only some of the applications. Gel electrophoresis is used in DNA fingerprintingwhich has applications in forensics. Certain DNA segments are characteristic and vary from person to person. These segments are cut at recognition sites by restriction endonuclease enzymes and run on gel by electrophoresis. The position and number of bands on each lane of gel is characteristic of the DNA sample and considered as the genetic "fingerprint" of that sample.

Next Section Gel Electrophoresis

References