Re: Why is agarose used in electrophoresis instead of agar ?

As agarose is nothing but agar, with only agaropectin removed from it, I was wondering why do we not use Agar for electrophoresis instead of agarose?
Furthermore, how does addition of water instead of buffer (which only acts to adjust the pH) lead to melting of casted gel in electrophoresis ?

Hi Tushar,

I think I will answer your second question first. I'm afraid that your statement that electrophoresis buffer only acts to adjust the pH of your agarose gel is incorrect. The primary purpose of the electrophoresis buffer is to provide an electrolyte-rich solution to conduct an electric current across the gel. Electrolytic conductivity in an electrophoretic cell is essentially the inverse of the resistance in an electrical circuit.

You may recall Ohm's Law, which relates current (I) to voltage potential (V) and electrical resistance (R) as V= I*R, and which can be rewritten as R = V/I. Electrolytic conductivity (κ) also relates directly to voltage potential and current, essentially as κ = I/V. Conductivity is measured in units of siemens / meter (S/m), and deionized water (such as you will use to make your solutions in lab) has a conductivity of about 5 μS/m, whereas tap water has a conductivity of 5 mS/m to 50 mS/m and sea-water has a conductivity of about 5 S/m. So, a 2.5% salt solution (sea water) has a million-fold greater conductivity than deionized water.

Since κ = I/V, I = V*κ. So, by using a buffer with a higher conductivity than deionized water, you can run your gel at a lower voltage and get the same current. Since κ α 1/R, a higher conductivity means less resistance and therefore less heat. This is important, because agarose will melt at 100 degrees C, and both you and I have had personal experience with inappropriately set-up gels overheating and partially melting during electrophoresis.

Now, as to the issue of agarose versus agaropectin, you should remember the purpose of running an agarose gel. I've written this answer about the history of the application of agar in the life sciences, which you might read as background. The thing that makes agarose so appealing for electrophoresis is that it does not interact with the buffer, the current or the biomolecules moving through it. Agarose is a polysaccharide polymer of disaccharide monomers with a neutral charge. This makes for a very uniform electrophoresis medium; by controlling the length of agarose molecules and the concentration of agarose in the gel, you can control the pore-size and density of the agarose matrix. This allows you to different types of agarose gels to separate different molecules on the basis of size; an agarose gel that can separate amplified DNA molecules that are a few hundred nucleotides in length is different from an agarose gel that can separate large pieces of chromosomes.

Agaropectin is not a single uniform uncharged molecule. It is actually a collection of many different and more complex polysaccharide molecules, which have charged (acidic) side-groups, primarily sulfate groups and acetyl groups from pyruvate. These acidic side groups prevent agaropectin from forming a uniform structure the way that agarose does; for example, calcium bridges can form between the amylopectin sulfate groups, resulting in large agaropectin structures. As a result, the structure of a gel containing agaropectin is not uniform, which makes it very bad for separating biomolecules. Because it has these charged side-groups, agaropectin can interact with the buffer, the current and the other biomolecules you are trying to electrophorese. This means that you can't reliably separate biomolecules in a pure agar gel.

Finally, in addition to agarsose and agaropectin, agar contains other contaminants (vitamins, amino-acids, salts and "inhibitory substances" according to Ryan et al., 1943 and Ryan, 1950) that make it a poor substrate for electrophoresis. So, agarose is not, "nothing but agar with the agaropectin removed." It is a highly purified and well characterized molecular biology reagent!

References
Imeson, Alan. Chapter 3. Agar. In, Food Stabilisers, Thickeners and Gelling Agents. Alan Imeson, ed. Wiley Blackwell. 2010. pp. 368.

Araki, C., 1937. Acetylation of agar-like substance of Gelidium mansii L. J.Chem.Soc.Japan, 58:1338-50.

Ryan, F. J. 1950. Selected methods of Neurospora genetics. Methods Med. Res. 3: 51-75.

Ryan, F. J., G. W. Beadle, and E. L. Tatum. 1943. The tube method of measuring the growth rate of Neurospora. Am. J. Bot. 30: 784-799.