ELECTROPHOREITIC TECHNIQUES: PAGE, western blotting, immunoblotting Electrophoresis refers to the migration of charged particles in an electric field.

Many important biological molecules for example proteins possess ionisable groups. Thus under the influence of an electric field such charged particles will migrate either to cathode or the anode depending on the nature of their charge. Thus, migration is proportional to potential gradient (E) x the charge on the particle (q). However, migration of molecules is also determined by frictional forces (f). Frictional forces include different characteristics of the molecule or matrix e.g. size or shape of the molecule, viscosity of the buffer or pore size of the matrix. As a result, the velocity in the field (v) can be expressed as v = E.q/f and v/E = electrophoretic mobility () So, molecules with different charges will separate and molecules that may have the same size but different size can also separate. The apparatus used for electrophoresis consists of a power pack and an electrophoresis unit. Electrophoresis units can run either vertically or horizontally. Vertical units are generally used for proteins and DNA sequencing. Horizontal units are used for DNA and RNA analysis. The power pack supplies constant current or voltage. The matrix used for electrophoresis consists of a porous mechanical support. The vast majority of electrophoretic techniques involve either agarose or polyacrylamide gel. Agarose is a linear polysaccharide made up of alternating repeats of galactose and 3,6 anhydrogalactose. Concentrations of 0.5% to 2.5% are used depending on pore size required. The gel consists of a cross linked structure due to the presence of both inter and intramolecular hydrogen bonding between and within the agarose chains. This cross linkage gives the gel good anticonvectional properties. The pore size in the gel is determined by the initial concentration of agarose, i.e. large pores are formed from low concentrations and smaller pore sizes are formed from higher concentrations. Agarose gels are generally used to separate DNA and RNA on the basis of their size. The presence of a phosphate backbone means that there is a constant charge/mass ratio. Electrophoresis in polacrylamide gels is often referred to as PAGE. Cross linked polyacrylamide gels are formed from the polymerization of the acrylamide monomer in smaller amounts of N,N-methylenebisacrylamide. Bisacrylamide is basically two acrylamide molecules linked by a methylene group and acts as a cross linking agent. The polymerization of acrylamide is initiated via free radicals by the addition of ammonium presulphate and the base TEMED. Like in agarose gel, the higher the concentration of acrylamide the smaller the pore size. Acrylamide gels can be made with a content of between 3% and 30% acrylamide. Gels of between 10% and 20% acrylamide are used in techniques such as SDSgel electrophoresis, where the smaller pore size introduces a sieving effect that contributes to the separation of proteins according to their size.

SDS-Page: Sodium dodecyl sulphate polyacrylamide gel electrophoresis is the most widely used method for analyzing protein mixtures qualitatively. It is particularly used for monitoring protein purification and because it is based on the separation of proteins according to size it can also be used to determine the relative molecular mass. Typically the separating gel used is a 15% polyacrylamide gel. This gel gives a pore size where proteins of Mr of 10,000 move through easily while proteins of Mr>100,000 can just enter the pores. A protein of higher Mr e.g. 150,000 would be unable to enter this 15% gel and would need a gel with larger pores. Thus, the choice of gel to be used depends on the size of the proteins being studied. SDS is an anionic detergent that denatures proteins. Samples to be run on SDS-PAGE are firstly boiled for five minutes in sample buffer containing -mercaptoethanol and SDS. The mercaptoethanol is a strong reducing agent that breaks the disulphide bridges present that are holding the protein tertiary structure together i.e. linearises the protein. The SDS binds to and denatures the protein by wrapping around the polypeptide backbone. The denatured proteins open up into a rod shaped structure with a series of negatively charged SDS molecules along the polypeptide chain. The SDS binds to all proteins at a constant ratio of approximately 1 SDS:2 amino acids. Because of this ratio, all proteins regardless of sequence are equally effected by the negative charge of the SDS and thus get same charge to mass ratio. Therefore, migration through the gel is proportional to molecular weight/size. The sample buffer also contains an ionisable tracking dye and surcrose or glycerol. The dye is usually bromophenol blue. This dye allows the electrophoretic run to be monitored. The presence of sugar gives the sample solution density thus allowing it to settle easily into the loading wells. A vertical gel apparatus is used for protein separation. The samples to be separated are not loaded directly into the main separating gel. Instead, the electrophoretic unit is a discontinuous gel system that consists of a stacking and resolving gel. The resolving gel is the main separating gel and is poured between the two glass plates and allowed to set. The stacking gel is then poured on top. The proteins are loaded into wells in this stacking gel. The purpose of the stacking gel is to concentrate the protein into a sharp band before it enters the resolving gel. Once all the samples are loaded, a current is passed through the gel. The stacking gel has a very large pore size which allows the proteins to move freely and concentrate at the top of the resolving gel. The negatively charged protein-SDS complexes move through the gel towards the anode. As they pass through the separating gel, the molecules separate according to their size. The smaller the protein the easier it can pass through the pores of the gel. While larger proteins are restricted by frictional resistance due to sieving properties of the gel. The bromophenol dye is a small molecule so is not restricted. Once the dye reaches the bottom of the gel the current is turned off. The gel is removed from between the glass plates and shaken in an appropriate stain solution e.g. Coomassie Brilliant Blue (can detect bands of 100ng) and then washed in a destain solution. The destain solution removes unbound background dye so that stained proteins can be seen as visible blue bands on a clear background. Uses Determination of Mrthe Mr of a protein can be determined by comparing its mobility with those of known Mr run on the same gel. A calibration curve can be constructed by plotting a graph of distance moved against log Mr for each of the known proteins. Once the distance moved by the unknown protein is known, its log Mr and thus Mr can be determined from the calibration curve. Used after each step of a purification protocol to assess purity. A pure protein should give a single band in SDS-PAGE.

Western Blotting & immunoblotting: In the electrophoresis process, PAGE achieves fractionation of a protein mixture. Western Blotting and Immunoblotting make use of this fractionation to further examine individual proteins. Firstly, the pattern of separated proteins is transferred/blotted from the gel onto a sheet of nitrocellulose paper. This process is known as Western blotting. The transfer of the proteins to the paper is achieved by a technique known as electroblotting. The gel and nitrocellulose are compressed and immersed in buffer between two electrodes. A current is passed at right angles and the proteins move from the gel onto the nitrocellulose sheet, to generate a replica of the protein gel that is easier to probe. The proteins are bound to the sheet by strong hydrophobic interactions. The nitrocellulose with the transferred protein is known as a blot. Once transferred, the proteins can be examined further. The blot is probed, usually with an antibody to detect a specific protein. Firstly the blot is incubated in a protein solution e.g. bovine serum albumin which will block all remaining hydrophobic binding sites. It is then incubated in a dilution of an antiserum/primary antibody specific for the protein of interest. If it detects the antigen of interest, this antibody will bind to the blot. The blot is washed to remove excess antibody. In order to visualize this interaction, the blot is incubated in a solution of secondary antibody, which is directed against the primary antibody. This secondary antibody is labeled so that the interaction of the two antibodies can be visualized. One of the most common detection methods is to use an enzyme-linked secondary antibody. After incubation with this secondary antibody, the blot is incubated in a enzyme-substrate solution. The enzyme will convert the substrate into an insoluble coloured product that is precipitated onto the nitrocellulose. The presence of the coloured band is indicative of the protein of interest.