PAGE ELECTROPHORESIS PRESENTED BY: KOUSHIK DAS Roll no.10 S.I.F, C.U.S.A.T-16
What is Electrophoresis ? Electrophoresis is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles.
GEL MATERIALS- USED INELECTROPHORESISAt first stage it is done in the freesolution called “Boundary Technique”.Filter paper, cellulose acetate strips etc.are used. But that Gel system isintroduced for electrophoresis.Generally starch gel system was used,replaced by agarose or polyacrylamidegels.
DIFFERENT TYPES OF GEL1. Agarose gel2. Polyacrylamide gel3. SDS (sodium dodecyl sulphate)4. Native PAGE5. Gradient gel
AGAROSE GELAgarose is one of the component ofagar obtained from certain seaweeds.Agarose is usually used atconcentration between 1% and 3%.
Preparation of Agarose GelAgarose gels- formed by suspending dry agarose inaqueous buffer, then boiling the mixture until a clearsolution is formed.This is poured and allowed to cool in room temperatureto form a gel.The pore size is controlled by the concentration ofagarose.Although essentially free of charges, that are alternatingsugar residues get substituted with carboxyl, methoxyl,pyruvate or sulphate to varying degrees.Therefore agarose is sold in different purity gradesbased on the sulphate concentration.
Uses of Agarose Gel Agarose are used for electrophoresisseparation of both proteins and nucleic acid.The pore size of 1% agarose gel are largerelatively to the size of proteins.Hence, agarose gels are used inimmunoelectrophoresis or isoelectricfocussing. Availability of low melting temperatureagarose(62-65ᵒC) allows these gel to bereliquified by heating to 65ᵒC and thus DNAsamples may be recovered.
POLYACRYLAMIDE GELSPreparation:Cross-linked polyacrylamide gels areformed from the polymerization ofacrylamide monomer in the presence ofsmall amount of N,N- methylenebisacrylamide.Bisacrylamide is essentially twoacrylamide molecules liked by a mehylenegroup used as cross-linked agent.
Preparation (continued): Acrylamide molecules is polymerized in a head to tail and bisacrylamide molecule. Polymerization of acrylamide through free radical catalysis and is initiated by the addition of ammonium persulphate and the base N,N,N,N- tetramethylamide (catalyst) Polymerization of acrylamide can be photopolymerization where ammonium persulphate and TEMED are replaced by “Riboflavin”
Preparation (continues):The gel is placed under bright light for 2-3 h.Photodecomposition of riboflavin generate free radicals that initiate polymerization.Total percentage of acrylamide usually 3% to 30%Pore size can varied by changing the concentration of both acrylamide and bisacrylamide.
SDS-PAGE(Sodium dodecyl sulphate)For DNA stacking SDS- polyacrylamide gels is used.Gels between 10% and 20% are used for SDS- gel electrophoresis where the small pore size act as a sieve and separate the proteins according to their molecular weight.Gels are formed in two forms.Tube gels and cylindrical rods of polyacrylamide.
Uses of SDS-PAGESDS is an anionic detergent which binds to and denature most protein1.4 gm of SDS binds per gm of proteinsSDS denature proteins a rod shapeThe length of which depends on the mass of proteinsSDS denature the proteins migrate through the gel according to there mass.
NATIVE -PAGE In native page protein are not denatured and electrophoresis is carried out in a variety of buffer system Depending on isoelectric point of protein and its various pH Protein separate according to their mobility and sieving effect of gel Its aim to detect a particular protein
GRADIENT GELSThis is a polyacrylamide gel systemBut in gradient gel system is not uniform poreThe acrylamide concentration varies from 5% at the top to 25% at the bottom of the gelTherefore as the sample moves down ,the pore size decreases
ADVANTAGES1. Protein with much greater range of molecular weight are separated very easily2. In a complex mixture , very low molecular weight protein travel freely through the gel3. Large protein separate immediately due to the sieving effects of gel4. Two protein of similar size but slightly different molecular weights will separate
USE OFELECTROPHORESISMolecular BiologyMedicinesQuality controlPurity tests Fluorescence checks Phenol tests KOH for white vs. redForensics lab.Genetics