In 1948, a Swedish Physical Biochemist, Arne Tiselius was awarded the Nobel prize in chemistry for the discovery of proteins in blood serum and for studying the properties of proteins through electrophoresis. Till now, electrophoresis continues to be an important technique to identify and characterize biological macromolecules. Amino acids, peptides, proteins and nucleic acids possess ionisable groups and can be made to exist in solution as cations or anions. When a mixture of these components is subjected to an electric field, they migrate differently and can be separated.
Electrophoresis is defined as the migration of charged particles, under the influence of an electric field at a definite pH. In a mixture of proteins, each protein with its electrical charge will move differently in an electric field. This electrophoretic mobility depends on the pH of the medium, strength of the field, net charge of the molecule and size/shape of the molecule. Electrophoresis is used for the analysis of large molecules (proteins and nucleic acids) and simpler charged molecules (peptide, simpler ions).
The following are different types or electrophoresis.
1. Paper electrophoresis
2. Cellulose acetate electrophoresis
3. Capillary electrophoresis
4. Gel electrophoresis
Agarose gel electrophoresis, Polyacrylamide Gel Electrophoresis (SDS PAGE, Native PAGE and two- dimensional electrophoresis).
Paper electrophoresis is an inexpensive method and requires only micro-quantities of protein. The apparatus consists of two troughs to accommodate a buffer through which an electric current is applied (Fig.10.9). Paper is a popular support medium as it is easy to handle, less expensive and is readily available. Paper contains 98% of cellulose. Paper electrophoresis has potential limitations. The greatest problem is the thickness and large pore size of the paper. The separation of proteins by paper electrophoresis takes longer time which limits its use.
Polyacrylamide gel is prepared from acrylamide and bis-acrylamide in a suitable buffer. Polymerization of Acrylamide and bisacrylamide is achieved by a free radical reaction promoted by N,N,N’,N’tetramethylethylenediamine (TEMED). This free radical process is initiated by Ammonium per sulfate (APS) used in gel. Acrylamide and bisacrylamide monomers are weak neurotoxin whereas, the polymerised polyacrylamide is non-toxic. While handling acrylamide solutions, care should be exercised and spectacles, gloves and mask should be worn.
Sodium dodecyl Sulphate Polyacrylamide Gel Electrophoresis(SDS-PAGE) is an electrophoretic technique very commonly used in Biochemistry, Molecular biology and forensic science. This technique was first described by Laemmli in the year 1970 and till now dominates in scientific research.
Electrophoresis apparatus: The electrophoretic apparatus consists of a reservoir tank to fill running buffer, transparent insulating cover, gel plates, spacers and gel comb to form wells. Platinum electrodes provide even current with the help of a regulated power pack. The gel is packed in-between two glass plates with the help of spacers. Clear wells are obtained using comb. Samples are layered in the little slots cut in the top of the gel slab using gel comb. Buffer is cautiously layered over the samples, and a voltage is applied to the gel using power pack for a period of usually 1-3 h. The proteins migrate in the gel depending upon their electrophoretic mobility, which is dependent on the size.
Protein samples to be run on SDS-PAGE are added to sample solubilizing buffer containing beta mercaptoethanol (disrupt disulphide bridges), SDS, glycerol (to make the solution denser and enable proteins to sink in the gel) and bromophenol blue (tracking dye).
SDS -PAGE contain resolving gel, used for separation of proteins and stacking gels for concentrating the proteins prior to entry into resolving gel. Sodium dodecyl sulphate (SDS) is an anionic detergent, which binds to proteins, and provides a constant negative charge per unit mass. Protein-SDS complexes will therefore move towards the anode during electrophoresis and their mobilities are inversely proportional to the log of their molecular weights. Since the SDS impart proteins have the same charge per unit length, all proteins travel with the same mobility. However, as the mixture of proteins pass through the resolving gel, the proteins separate, owing to the molecular sieving properties of the gel. The smaller proteins move fast as they can pass through the pores of the gel. But larger proteins move slowly since they are retarded by frictional resistance due to sieving effect of the gels. When the dye reaches the bottom of the gel, the current is turned off. After electrophoresis, the gel is carefully removed from the glass plate, immersed in buffer and stained with appropriate stain solution (Fig.10.10)
Protein staining: Proteins can be detected using Coomassie Brilliant Blue G250 (CBB) solution. CBB dye stains protein with a detection limit of 40μg. For proteins of less quantity, another sensitive detection known as silver staining (1-5ng detection limit), can be performed.
Applications : SDS–PAGE is used to determine the molecular weight of proteins. To achieve this, a standard mixture of proteins of various molecular weight (molecular weight ladder) was added for direct comparison of migration distance. The molecular weights around 15-200kDa can be analyzed in this manner.
Two dimensional gel electrophoresis was introduced by O’Farell in the year 1975. It is a combination of two techniques, iso electric focusing and SDS-PAGE. Iso-electric focusing is an electrophoretic technique where proteins are separated based on their iso-electric point (pI). pI is the pH at which the amino acid does not migrate in an electric field (zwitterion form). When a gradient of pH is applied to a gel, and electric field applied to it, one end becomes more positive than the other. Relatively at all pH other than its iso electric point, proteins have a charge (positive or negative) and will be pulled to the opposite side of the gel. In two dimensional electrophoresis, proteins are separated based on isoelectric point and molecular mass. To accomplish this, proteins are first separated by isoelectric focusing where they are separated by their respective isoelectric point. Second dimension of separation is achieved through SDS-PAGE, where proteins are separated according to their molecular weight. Each spot on the resulting 2D gel correspond to single protein species present in the sample (Fig.10.11)
Applications : SDS–PAGE can be used to determine the molecular weight of proteins. To achieve this, a standard mixture of proteins of various molecular weight (molecular weight ladder) was added for direct comparison of migration distance. The molecular weights around 15-200kDa can be analyzed in this manner. Another application of SDS PAGE is to check the purity of a protein sample. Presence of a single band denote the protein sample is pure.
Agarose is one of the several components that can be separated from agar. The major source of agar is certain species of sea weed. Agarose is a linear polymer made up of alternating units of galactose and 3,6- anhydrogalactose. Agarose gels are completely transparent when cooled to room temperature.
In Agarose gel electrophoresis, DNA or RNA molecules can be separated based on their size. This is achieved by the movement of negatively charged nucleic acid molecules through an agarose matrix in a horizontal electrophoresis. Molecules with smaller size move faster and migrate farther as compared to longer ones. The distance between DNA or RNA bands of a given length is determined by the percentage of agarose in the gel.