Sample Prep for Denaturing PAGE of DNA
DNA samples for denaturing gel electrophoresis must be denatured prior to loading, to avoid time dependent denaturation artifacts on the gel. This is usually carried out by diluting the sample into 95% formamide and heating to 95°C (see the Dideoxy Sequencing (Taq Polymerase) Protocol for a formula for the loading buffer). Loading the proper amount…
Read MoreDenaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
The denaturation of DNA by urea. The electrophoretic analysis of single-stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form…
Read MoreHorizontal and Vertical Gel Systems – Vertical Tube Gels
Tube gels were used frequently in the development of gel electrophoresis. Although they are still used for some applications (most notably for isoelectric focusing as part of 2D electrophoresis), tube gels have been superseded by slab gels for most applications. Tube gels are cast (as the name implies) in glass tubes of 1-3mm diameter. The…
Read MoreHorizontal and Vertical Gel Systems – The Vertical Slab Gel System
The vertical gel electrophoresis apparatus. The gel is clamped into the apparatus so that the lower end is immersed in the lower buffer chamber, and the upper end forms one wall of the upper chamber. The gel provides the only electrical connection between the two buffer chambers. Cooling is provided by a metal heat sink…
Read MoreHorizontal and Vertical Gel Systems – The Horizontal Gel System
A gel electrophoresis apparatus must allow the researcher to maintain a uniform electric field across the gel, provide cooling to prevent thermal artifacts, and allow access to the gel for sample loading and monitoring the run. Two types of apparatus are in common use: vertical and horizontal. Vertical gel systems are further subdivided into slab…
Read MoreBuffer 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…
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