What properties does gel electrophoresis separate molecules?

Answer 1

Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. The direction of movement is affected by the charge of the molecules, and the rate of movement is affected by their size and shape, the density of the gel, and the strength of the electrical field.

DNA is a negatively charged molecule, so it will move toward the positive pole of the gel when a current is applied. When DNA has been cut by restriction enzymes, the different-sized fragments will migrate at different rates. Because the smallest fragments move the most quickly, they will migrate the farthest during the time the current is on. Keep in mind that the length of each fragment is measured in number of DNA base pairs.

Answer 2

This question is really dependent on what conditions you are going to run your protein under. Gels will separate proteins based on their charge/mass ratio. The more charged a protein is, the more attracted it will be to the electrode and consequently the faster it will migrate; likewise, the smaller a protein is, the easier it can slip through the gel pores and the faster it will migrate. However, it is possible to separate based solely on one characteristic or another.

If you run the protein after denaturing it with sodium dodecyl sulfate (SDS), the gel will separate the proteins based solely on molecular weight. This is because SDS denatures and then coats the proteins in fairly uniform amounts of negative charge; this uniform charge coat eliminates the proteins' variations in native charge, leaving molecular weight as the only variable by which to separate the proteins.

If you would prefer to separate proteins another way, you can separate them by their isoelectric point (pI). Proteins have various amounts of charge depending on what pH they are at. This property is often exploited to find out at which pH the protein has zero charge; this point is called their isoelectric point or pI. The gel is loaded into a chamber that contains a buffer that is under a pH gradient of known range (3.5-9.5, 6-8, etc.). The proteins will start off with some amount of charge and migrate towards the electrode that attracts them; their movement will cease when they reach their pI, as they will no longer be charged and therefore no longer be affected by the electric field.

However, the concentration of agarose also matters. Different molecules will behave differently in agarose, migrating more due to either weight or charge (or a mix of the two). In this case, it is best to search the literature to see if previous work has been done to show which behavior is dominant for the type of molecule that you are interested in.

Normally you would not separate protein on an agarose gel - you would use a SDS-PAGE (sodium dodecyl sulphate - polyacrylamide gel electrophoresis). The reasons for this are three fold. Firstly, the matrix that agarose forms is quite large and proteins are relatively small so that it would be hard to resolve proteins on the gel (you are more likely to just run them off the end). Acrylamide, which forms smaller pores is more suited to proteins. Secondly, as the answer above correctly points out, proteins run according to their molecular weight and their charge so SDS is used to negatively coat the protein thus allowing you to separate the protein simply by size (molecular weight). However, agarose gels are not usually run in conditions containing SDS and its not possible to get a protein to run into the gel unless it is negatively charged. It is possible to run proteins complexed with DNA as the DNA carries the net negative charge. This brings us to the third point. Proteins often form complexes, with themsleves, with DNA and with other proteins. The conditions that agarose gels are run under are not strong enough to break up these complexes or unfold the proteins so that proteins may run anomolously within the gel. Acrylamide gels containing SDS and a reducing agent such as DTT are much more suited and are used much more commonly.

Answer 3

Gel electrophoresis separates molecules based on their size and charge. Smaller molecules move faster through the gel than larger molecules, and molecules with a negative charge will move towards the positive electrode. This technique is commonly used to separate DNA, RNA, and proteins for analysis.

Answer 4

shape, charge, and size

Answer 5

DNA fragments have different lengths