Electrophoresis Definitions More

Electrophoresis Reagents

Agarose A large pore polymer used as a gel medium in electrophoresis of large molecules, particularly nucleic acids
Amino acids The building blocks of proteins. The twenty alpha-amino acids are used to synthesize proteins in all living systems. Some of these amino acids have charged side chains which can contribute to the charge behavior of proteins during electrophoresis.
Anode The positively charged electrode that attracts negatively charged proteins
Buffer A weak acid or base that resists change in pH
Denaturation A process by which the non-covalent bonds (hydrogen bonds, electrostatic forces, van der Waals forces) of proteins are broken thereby unfolding the protein into a random coil. SDS is used in electrophoresis to denature proteins.
DTT Dithiothreitol is a reducing agent used to break the disulfide bonds between
between cysteine residues in polypeptides.
Capillary Electrophoresis A technique where an electrical field is applied to either end of a capillary (a small diameter silicon tube). Molecules in the capillary then migrate based on their size and charge.
Cathode The negatively charged node that attracts positively charged proteins
Convection currents Naturally produced currents due to the movement of warm air. These currents affect the electrophoresis because the sample is constantly moving and therefore it cannot be guaranteed that all the polypeptide chains are completely separated from each other.
Electrophoresis A process by which proteins are separated based on their mobility in a gel medium
Glycoproteins Proteins with a covalently bonded carbohydrate attached
Lipoproteins Proteins bonded to a lipid
Mercaptoethanol A reducing agent
Molecular weight The sum of the atomic weights in a molecule which is expressed in amus (atomic mass units). The molecular weight of a molecule is directly related to its migration distance in an electrophoresis gel, the smaller the molecular weight the farther the polypeptide migrates, and the larger the molecular weight the less the polypeptide migrates
Native The native conformation is the original conformation of a protein molecule
Polyacrylamide Used as a gel medium in electrophoresis because it acts as a friction force on the migrating polypeptide chains
Primary structure A straight chain sequence of amino acids
Protein Constructed from polypeptides (sequences of 20 amino acids) which are folded/coiled into unique conformations
Random coil A random or irregularly bent/folded section of a polypeptie chain with a secondary structure. This irregularity provides a means for the polypeptide chain to bend back on itself.
Quaternary structure A globular protein created from two or more polypeptide chains bonded together
Reducing agents Substances such as DTT and mercaptoethanol which reduce a protein to its primary structure.
Relative migrationRm The amount a polypeptide chain moves in relation to the tracking dye. Also called the relative mobility, it is calculated by dividing the distance the polypeptide migrates by the distance the tracking dye migrates.
SDS Sodium dodecyl sulfate
Secondary structure Polypeptide chains with folds/coils due to hydrogen bonding between CO and NH groups
Sodium dodecyl sulfate

A detergent used to denature proteins thereby unfolding the protein to its primary structure

Subunits Polypeptides which make up the quarternary structure of a protein
Tertiary structure A polypeptide chain with folds/coils as well as bonding between side chains creating a 3D shape as a result of hydrophobic interactions and disulfide bridges

Electrophoresis Theory

  Electrophoresis is a technique used to separate molecules based on their size and charge, according to the following equation where v = the rate (velocity) of migration, E is the strength of the electrical field, z is the charge on the molecule and f is the frictional force on the molecule

where v = the rate (velocity) of migration, E is the strength of the electrical field, z is the charge on the molecule and f is the frictional force on the molecule. The frictional force can be defined as

where h is the viscosity of the medium and r is the stokes radius of the molecule.

In zonal electrophoresis, cations in solution migrate toward either the cathode (negatively charged) whereas anions migrate toward the anode (positively charged) when an electrical field is applied. The principles of zonal electrophoresis are used mainly in capillary electrophoresis these days.

In gel electrophoresis, a matrix consisting of either polyacrylamide (for proteins and small nucleic acids) or agarose (for larger nucleic acids) is prepared. A polyacrylamide gel contains long linear polymers of acrylamide that are cross-linked to each other using bis-acrylamide. Samples are applied in sample wells at the cathodic end of the matrix. When an electric field is applied, negatively charged species migrate toward the anode.

The gel serves two purposes. It serves to diffuse convective currents that would result in localized heating in the matrix, which would result in irregular migration patterns. The gel also creates a molecular sieve that enhances the separation based on molecular weight.

What does this applet do?

This applet simulates the process of electrophoesis on proteins. After separation the applet will plot the result and determine the experimental molecular weight of the unknowns. The experimental molecular weight will be calculated from the relative migration of the unknown as plotted on the graph of the knowns.

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