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PAGE ELECTROPHORESIS PRESENTED BY: KOUSHIK DAS Roll no.10 S.I.F, C.U.S.A.T-16

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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 IN

ELECTROPHORESIS

At 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 GEL

1. Agarose gel

2. Polyacrylamide gel

3. SDS (sodium dodecyl sulphate)

4. Native PAGE

5. Gradient gel

AGAROSE GEL

Agarose 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 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 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-PAGE

SDS 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 system

But 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 Biology

Medicines

Quality control

Purity testsFluorescence checks

Phenol tests

KOH for white vs. red

Forensics lab.

Genetics