Form and Function
Agarose can be used for electrophoretic separation in agarose gel electrophoresis or for column-based gel filtration chromatography. Agarose gel electrophoresis is a method used to separate DNA or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field. Shorter molecules move faster and migrate farther than longer ones.
Figure 1. Structure of agarose polymer.
1. Melting/Gelling Temperature: Agarose exhibits a high hysteresis (difference between melting and gelling temperatures) making it ideal for separations such as electrophoresis and chromatography. The gelling temperature ranges from 32 - 45°C, and the melting temperature range is normally 80 - 95°C depending on the type of Agarose preparation used. Melting and gelling temperatures can be changed by methylation, as well as alkylation and hydroxyalkylation of the polymer chain.
2. Gel Strength: Gel strength is particularly important when gels must be handled or blotted after electrophoresis. pH and other additives can effect the gel strength of Agarose; in particular, sulfate salts and the sulfate content in the agarose molecules reduce the strength of the gel.
3. Sulphate Content: Sulfate content may be used as an indicator of purity, since sulfate is the major ionic group present.
4. Electroendosmosis (EEO): Electroendosmosis (EEO) is one of the most important characteristics to consider when choosing an agarose to meet your electrophoretic needs. EEO is the movement of non-charged molecules through a medium toward the cathode during electrophoresis. If agarose is the medium, ions such as ester sulfate and pyruvate groups impart a net negative charge to the agarose. Though the gel itself cannot move, counter-ions and water associated with the sulfate and pyruvate groups move toward the cathode. As water migrates with the counter-ions, neutral molecules, which normally would not migrate, are pulled along with the water. This movement of molecules towards the cathode is known to slow the separation of DNA, so the lower the EEO, the faster DNA will migrate. In most electrophoretic procedures, EEO is an unwelcome side-effect. Just as the mobility of a charged molecule is a direct function of the voltage gradient, so is EEO. Additionally, lower EEO helps improve the resolution of DNA and RNA as their migration is determined only by their size, not by their charge.
How to measure EEO:
EEO is measured by subjecting a mixture of dextran and albumin to electrophoresis, then visualizing them, and measuring their respective distances from the origin. The amount of EEO (-Mr) is calculated by dividing the migration distance of the neutral dextran (OD) by the sum of the migration distances of the dextran and the albumin (OD + OA).
-Mr = OD/(OD + OA)
Type of Agarose -Mr range (Relative Migration Distance)
Applications of Agarose Gel Electrophoresis:
Agarose gel has the advantages that the gel is easily poured and it does not denature the samples. The samples can also be recovered. The disadvantages are that gels can melt during electrophoresis and different forms of genetic material may run in unpredictable forms.
Factors affecting migration
Percent Agarose and Resolution Limits
Recommended Usage (TBE):