What is the relationship between the DNA molecular weight and the distance Travelled by DNA fragments from the well?

Gel electrophoresis is used to characterize one of the most basic properties - molecular mass - of both polynucleotides and polypeptides. Gel electrophoresis can also be used to determine: (1) the purity of these samples, (2) heterogeneity/extent of degradation, and (3) subunit composition.

The most common gel electrophoresis materials for DNA molecules is agarose and acrylamide.

Acrylamide gels are useful for separation of small DNA fragments typically oligonucleotides <100 base pairs. These gels are usually of a low acrylamide concentration (<=6%) and contain the non-ionic denaturing agent Urea (6M). The denaturing agent prevents secondary structure formation in oligonucleotides and allows a relatively accurate determination of molecular mass.

Gel electrophoresis of proteins almost exclusively utilizes polyacrylamide. The acrylamide solution usually contains two components: acrylamide and bis acrylamide. A typical value for the acrylamide:bis ratio is 19:1. The bis acrylamide is essentially a cross-linking component of the acrylamide polymer. The total acrylamide concentration in the gel affects the migration of proteins through the matrix (as with the concentration of agarose).

Protein gels are usually performed under denaturing conditions in the presence of the detergent sodium dodecyl sulfate (SDS). The proteins are denatured by heat in the presence of SDS. The SDS binds, via hydrophobic interactions, to the proteins in an amount approximately proportional to the size of the protein. Due to the charged nature of the SDS molecule the proteins thus have a somewhat constant charge to mass ratio and migrate through the gel at a rate proportional to their molecular mass, The proteins migrate towards the anode.

Since the SDS treatment will dissociate non-covalent protein complexes, they may thus exhibit a much lower than expected molecular mass on SDS polyacrylamide gel electrophoresis (SDS PAGE). Protein PAGE gels are usually polymerized between two glass plates and run in the vertical direction.

Figure 3.1.4: Effect of SDS treatment

PAGE may also be run in the presence of reducing agents, such as b-mercaptoethanol (BME). BME is a reducing agent which will reduce any disulfide bonds (e.g. as exists between some pairs of cysteine residues in a protein). This helps to remove residual secondary structure in the SDS treated protein, but it may also allow the separation of polypeptide fragments from each other (i.e. their covalent interaction was entirely made up of one or more disulfide bonds). Thus, an apparently single protein may exhibit a set of small fragments under reducing PAGE conditions.

• Coomassie blue is a triphenylmethane textile dye which is able to stain proteins.
  • After a polyacrylamide gel is run it is usually "fixed" by placing in a 50% methanol/10 acetic acid solution for 30 minutes (to precipitate the proteins and prevent diffusion out of the gel).
  • The fixed gel is then soaked in a methanol/acetic acid solution containing 2.5 gm/liter of Coosmassie blue.
  • Destaining of the background gel is accomplished by soaking in changes of 10% methanol/7 % acetic acid. This method can typically detect protein samples of 0.1 ug or greater.

• Silver staining is a method which utilizes a silver nitrate solution to stain proteins in an acrylamide gel.
• The method is similar in nature to the use of silver in photographic plates.
• The sensitivity is approximately two orders of magnitude more sensitive than coomassie staining (i.e. one can detect approximately 1ng of protein).
• Due to its high sensitivity this method is usually used to determine the presence of trace contaminants in protein samples.