Electrophoresis Articles
Buffer Additives-Reducing Agents
Disulfide bonds between or within sample protein molecules can lead to the formation of aggregates as well as play a role in the binding of the subunits of many proteins. It is usually desirable to cleave disulfide linkages prior to the protein electrophoresis. For this reason, disulfide bond reducing agents, such as 2-mercaptoethanol or dithiothreitol,…
Read MoreBuffer Additives-Surfactants
A crucial initial step in the electrophoretic separation of proteins is the solubilization of the sample molecules. This is especially true if there are extensive nonpolar interactions. Although urea in high concentration was often employed in the past for this purpose, researchers now often have recourse to the use of nonionic, anionic, or cationic detergents.…
Read MoreBuffer Additives-Hydrogen Bonding Agents
In most forms of electrophoresis, the solution perfusing the gel matrix typically contains one or more substances in addition to the buffer salts. Serving the purpose of modifying the properties of sample molecules, these additives can be categorized as hydrogen bonding agents, surfactants, or reducing agents. Hydrogen bonding agents Urea or formamide can be introduced…
Read MoreIsotachophoresis
Isotachophoresis is a method of electrophoresis that employs the basic principles of the stacking gel phase of multiphasic systems discussed in the preceding section. Employing nonsieving media, often low percentage polyacrylamide, isotachophoresis in its simplest form can be thought of as a stacking gel alone, without the separation gel. The principles are the same. With…
Read MoreMultiphasic Buffer Systems
At the start of multiphasic SDS-PAGE protein electrophoresis, the anions are chloride (green) in the stacking and resolving gels and glycine (orange) in the tanks. Employing gel and buffer discontinuities to produce sharp separation among sample components, multiphasic electrophoresis design can improve the resolution of electrophoresis (especially protein electrophoresis). The system employs a separating gel…
Read MoreHomogeneous Buffer Systems
In a homogeneous buffer system, the identity and concentration of buffer components are the same in the gel and the tanks. Most forms of DNA and RNA electrophoresis generally use homogeneous buffer systems. Electrophoresis of proteins is most often performed under multiphasic conditions, where tank and gel buffers differ. In a homogeneous system, the buffer…
Read MoreElectrophoresis Buffers-Choosing the Right Buffer
Several factors to consider when choosing a buffer include: 1) pKa value – A buffer should be chosen with a pKa that is very close to the desired pH, preferably within a half point. The buffer will have the greatest capacity both to absorb or release protons with the acid and the base form well…
Read MoreThe Polyacrylamide Matrix-Buffer Strength
The buffer system in electrophoresis controls the pH of the gel, preventing damage to sample molecules and, in certain cases, controlling the ionization state of the molecules. A second, though no less significant function, derives from the fact that the vast majority of current flowing through the electrophoresis gel is carried by the buffer ions.…
Read MoreElectrophoresis Buffers–The Henderson-Hasselbalch Equation
In its simplest form, a buffered solution contains a mixture of a weak acid and its conjugate base. The position of acid/base equilibrium is represented by the acid dissociation constant, Ka. This number is large if the acid is stronger and equilibrium tends toward dissociation. It is small for an equilibrium that tends toward proton…
Read MoreThe Agarose Matrix
Agarobiose is the basic unit of the agarose. A natural colloid extracted from seaweed, agarose is a linear polysaccharide made up of the repeating unit agarobiose, which consists of alternating units of 1,3-linked b-D-galactopyranose and 1,4-linked 3,6-anhydro-a-L-galactopyranose. Gels prepared from agarose have a substantially larger pore size than polyacrylamide gels. Agarose gels can be prepared…
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