Separation of Amino Acids by Gel Electrophoresis

Gel electrophoresis is used to characterize one of the most basic properties - molecular mass - of both polynucleotides and polypeptides. Here we will focus exclusively on gel electrophoresis of proteins Gel electrophoresis can be used to determine: the purity of a protein sample heterogeneity and extent of degradation of a protein sample subunit composition of a protein sample How does it work? The underlying principle of electrophoresis is the migration property of charged species within an electric field. Thus, it is the simple behavior of opposite charges attracting. An electric field is established across the electrodes of a power supply, and charged ions move in this electric field. Note that in such an apparatus, the word “ANODE“ refers to the positively charged electrode (the ANODE attracts ANIONS) and the word “CATHODE“ refers to the negatively charged electrode (the CATHODE attracts CATIONS). The cathode and anode terms are consistent with their redox reaction definitions in that reduction is subsequently occurring at the cathode and oxidation is occurring at the anode. Proteins are comprised of the 20 common amino acids, which include both negatively charged (i.e. acidic) side chains (e.g. aspartic acid, glutamic acid) and positively charged (i.e. basic) side chains (e.g. histidine, lysine and arginine). Thus, proteins can be charged, and will migrate in an electric field. At this point there are a couple of things to consider: 1) Any such separation is a non-equilibrium process. By this, we mean that if we let the process continue on until some equilibrium condition is met, all the anions will be on one electrode and all the cations will be on the other. It would be better to halt the separation process at some intermediate time point to permit achieve separation. 2) The other problem is that once the electric field is switched off, diffusion will cause the separated ions to move around (i.e. we will lose the separation we have tried to achieve). To solve this problem, the separation is not performed in solution, but within a matrix (i.e. a molecular mesh or network). The matrix provides a frictional component that resists diffusion. Furthermore, the friction of the matrix is an important factor in the rate of migration of the ions. 3) Note also that separation is achieved by initial application of the sample within a narrow zone (band). If the sample is initially dispersed, although the ions will move, they won’t be neatly separated. Factors that influence the rate of electrophoresis migration (Rf) Three factors affect the rate of migration: · Strength of the electric field, E (directly proportional to migration rate) · Charge on the ionic species, q (directly proportional to migration rate) · Frictional coefficient of the support matrix, f (inversely proportional to migration rate) Rf α qE/f These factors can be varied in the following way: · The strength of the field is a function of the voltage of the power supply. Thus, we can vary the voltage directly. In a related issue, the voltage is proportional to the resistance across the electrodes. Current comes into play here also, but in short, it is difficult to achieve high voltage across the electrodes if the resistance is low. The resistance of a solution is inversely proportional to the ionic strength (i.e. concentration of ions). Thus, with high salt concentrations, the resistance is low, it is difficult to achieve high voltage, and the migration rate will decrease. #GelElectrophoresis #proteins #aminoAcids #zwitterions #laboratoryQuiz #protein #agarose #DNABands #usingAGelElectrophoresisMachine #DNAFingerprinting #howDoesAGelElectrophoresisMachineWork #biology #science #restrictionEnzymes #guppies #DNAFragments #howDNAMovesThroughGelElectrophoresis #DNALadder #semilogGraph #purposeOfGelElectrophoresis #biotechnology #highSchool #stepsInElectrophoresis #NikolaysGeneticsLessons