Electrophoresis PAGE Dr Gihan Gawish

Types of Gels The most common types of gels are: Starch gels: seldom used nowadays Agarose gels: for separation of nucleic acids and large proteins Polyacrylamide gels: for separation of most proteins and small nucleic acids Dr Gihan Gawish

Polyacrylamide Gel Electrophoresis (PAGE) PAGE can be classified according the separation conditions into: 1- Native-PAGE: Separation is based upon charge, size, and shape of macromolecules. Useful for separation and/or purification of mixture of proteins This was the original mode of electrophoresis (introduced in 1930s, Nobel Prize 1948). 2- Denatured-PAGE or SDS-PAGE Separation is based upon the molecular weight of proteins. The most common method for determining MW of proteins Very useful for checking purity of protein samples Dr Gihan Gawish

PAGE 3- Isoelectric Focusing-PAGE Separation of basis of pI, not MW Recently, became popular as a part of proteomic techniques PAGE can also be classified according to the physical shape of the gel: slab (most common) or tubes Continuous, discontinuous, stacked, or gradient gel One dimensional or two-dimensional electrophoresis Dr Gihan Gawish

SDS-PAGE Biopolymers such as proteins and nucleic acids are folded into compact structures They held together by a variety of non- covalent, ionic interactions such as hydrogen bonding salt bridges. Dr Gihan Gawish

SDS-PAGE The electrophoretic mobility of the denaturated molecule will be changed, compared to that in non denaturating conditions It migrates as an unstructured monomer through the electrical field. Dr Gihan Gawish

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE, is a technique widely used to separate proteins according to their electrophoretic mobility The SDS gel electrophoresis of samples having: identical charge to mass ratios results in fractionation by size Dr Gihan Gawish

SDS-PAGE disrupting its compact folded structure. Dr Gihan Gawish The detergent sodium dodecyl sulphate (SDS) consists of: hydrophobic 12-carbon chain polar sulphated head Hydrophobic chain can Intercalate into Hydrophobic parts of the protein by detergent action disrupting its compact folded structure. Dr Gihan Gawish

Dr Gihan Gawish

SDS-PAGE SDS is highly charged (-) binds to proteins in proportion to their molecular weight SDS molecules exert strong internal repulsive forces tend to stretch out the random coil protein into a rod-like shape All proteins are made to look virtually the same rod diameter but with lengths that are proportional to the molecular weight of the protein. Dr Gihan Gawish

SDS-PAGE: Procedure The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non– disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide. Dr Gihan Gawish

SDS page Dr Gihan Gawish

SDS PAGE in practice Denatured SDS-protein mixture with a colored dye or stain added for tracking is loaded at the top of a slab or tube of a gel (typically polyacrylamide) Electric field imposed within the gel using electrodes attached to a power supply Proteins and dye migrate down the gel at a constant rate that depends on the molecular weight of the protein Smaller proteins migrating faster Dr Gihan Gawish

SDS PAGE in practice Over a limited molecular weight range, the electrophoretic mobility of proteins is found to be proportional to the logarithm of their molecular weight Dr Gihan Gawish

SDS- PAGE Advantages Rapidly and cheaply measure molecular weights with an accuracy of about 5% determine trace amounts of impurities in a sample Dr Gihan Gawish

Preparative SDS-PAGE Samples are electrophoresed (native or SDS PAGE) through a cylindrical gel Bands pass through a thin frit within the elution chamber Isolated bands are drawn radially by a pump onto a fraction collector into discreet liquid fractions Dr Gihan Gawish

Dr Gihan Gawish

Isoelectric focusing-PAGE Isoelectric focusing is a technique for separating different molecules by their electric charge differences. The charge of molecule changes with the pH of its surroundings. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the negative electrolode. Dr Gihan Gawish

Isoelectric Focusing (IEF)-PAGE Employs a pH gradient extending the entire gel: (slabs or tubes) Carrier ampholytes are used to set up the pH gradient Protein sample is applied: no SDS. charges make proteins different Dr Gihan Gawish

IEF-PAGE At pH = pI, a protein will have no net charge  stop moving At any other pH in the gradient, the protein has either a positive charge (pH<pI) or negative charge (pH>pI) Runs requires higher voltages and longer periods of time, but gives resolution up ±0.001 pH Dr Gihan Gawish