PAGE ELECTROPHORESIS

PRESENTED BY: KOUSHIK DASRoll no.10S.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 IN ELECTROPHORESIS

At first stage it is done in the free solution called “Boundary Technique”. Filter paper, cellulose acetate strips etc. are used. But that Gel system is introduced for electrophoresis.

Generally starch gel system was used, replaced by agarose or polyacrylamide gels.

DIFFERENT TYPES OF GEL

1. Agarose gel2. Polyacrylamide gel3. SDS (sodium dodecyl sulphate)4. Native PAGE5. Gradient gel

AGAROSE GELAgarose is one of the component of agar obtained from certain seaweeds.

Agarose is usually used at concentration between 1% and 3%.

Preparation of Agarose Gel

Agarose gels- formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution is formed.This is poured and allowed to cool in room temperature to form a gel.The pore size is controlled by the concentration of agarose.Although essentially free of charges, that are alternating sugar residues get substituted with carboxyl, methoxyl, pyruvate or sulphate to varying degrees.Therefore agarose is sold in different purity grades based on the sulphate concentration.

Uses of Agarose Gel

Agarose are used for electrophoresis separation of both proteins and nucleic acid.The pore size of 1% agarose gel are large relatively to the size of proteins.Hence, agarose gels are used in immunoelectrophoresis or isoelectricfocussing. Availability of low melting temperature agarose(62-65ᵒC) allows these gel to be reliquified by heating to 65ᵒC and thus DNA samples may be recovered.

POLYACRYLAMIDE GELS

Preparation:Cross-linked polyacrylamide gels are formed from the polymerization of acrylamide monomer in the presence of small amount of N,N- methylene bisacrylamide.Bisacrylamide is essentially two acrylamide molecules liked by a mehylene group 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.

PAGE

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 protein

1.4 gm of SDS binds per gm of proteins

SDS denature proteins a rod shape

The length of which depends on the mass of proteins

SDS denature the proteins migrate through the gel according to there mass.

SDS- PAGE

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

NATIVE -PAGE

GRADIENT GELS

This is a polyacrylamide gel systemBut in gradient gel system is not

uniform pore The acrylamide concentration varies

from 5% at the top to 25% at the bottom of the gel

Therefore as the sample moves down ,the pore size decreases

ADVANTAGES

1. Protein with much greater range of molecular weight are separated very easily

2. In a complex mixture , very low molecular weight protein travel freely through the gel

3. Large protein separate immediately due to the sieving effects of gel

4. Two protein of similar size but slightly different molecular weights will separate as two ,close , sharp bands.

USE OF ELECTROPHORESISMolecular BiologyMedicinesQuality controlPurity tests

Fluorescence checksPhenol testsKOH for white vs. red

Forensics lab.Genetics

Thank you