Gel Electrophoresis, Principle, Types and Applications

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Gel Electrophoresis, Principle, Types and Applications

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Description of Module

Subject Name

Paper Name

Module Name/Title Gel Electrophoresis, Principle, Types and Applications

Dr. Vijaya Khader Dr. MC Varadaraj

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1. Objectives

1. To understand gel electrophoresis Principle

2. Types of Electrophoresis(for DNA, RNA, Proteins)

3. Chemistry of electrophoresis

4. Applications

2. Lay Out

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Gel Electrophoresis

Agarose SDS-PAGE 2D-E

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3. 1Description

When charged particles move in an electric field

Electrophoresis is most commonly used for biomolecule separation such as DNA, RNA or protein

May be used as a preparative technique prior to use of other methods such as RFLP, PCR, cloning, DNA

sequencing, or blotting

3.2 History

Father of electrophoresis: Arne Tiselius (Nobel Prize in 1948)

3.3 Principle

When we place any charged molecules in an electric field, they move toward the positive or

negative pole according to the charge they are having. Proteins do not have any net charge

whereas nucleic acids have a negative charge so they move towards the anode when electric

field is applied .

3.4 Factors Affecting gel Electrohoresis

Electrophoretic velocity depends on:

Inherent Factors

How much charge the particles have

What is the molecular weight

Secondary structures (i.e., its shape).

External Environment

pH of solution

Electric field

Solution viscosity

Temperature

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3.5 Gel

Proteins and nucleic acids are electrophoresed ( movement under the effect of electric current) in a

gel . Usually the gel is polymerized in the shape of a thin slab and have wells to load sample.

Agarose

Agarose is a polysaccharide extracted from seaweed(Gelidium) and contains many

agarobiose subunits

During solidification, agarose form a network of polymers and its pore sizes can be

determined by its concentration

It is usually used at concentrations of 0.5 to 2%.

Stiffer gel means the agarose concentration is higher

Heat agarose with buffer to prepare gel and after it cools down it is poured in to the tray

called as casting tray

These gels are not toxic unlike acrylamide gels

Range of separation in agarose gels is higher but resolving power is low.

By varying the concentration of agarose, we can separate 500 t0 4000 bp of DNA

3.6 Staining

Ethidium Bromide

Intercalation between base pairs of nucleic acids results in very strong binding

When EtBr is exposed to uv light, electrons in the aromatic ring of the ethidium molecule get

activated, which releases energy in the form of light

Stock prepared : 10mg/ml

Working concentration : 0.5 µg/mL.

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Precaution: EtBr is carcinogenic since it intercalates between base pairs of nucleic acids so not to

touch with bare hands. Always use gloves while handling stain and stained gels.

Other Available safe options for staining agarose gels:

There are other stains for DNA available in the market for agarose gels. Some of these stains comes

under name SYBR Gold, SYBR green, Crystal Violet and Methyl Blue. Methyl Blue and Crystal Violet do not

require exposure of the gel to uv light to see DNA bands, therefore reducing the chancesn of mutation. But

than they are less sensitive than EtBr.

SYBR gold and SYBR green have good sensitivity. These are ultra-violet light dependent dyes and are

less toxic than EtBr, but these are not very economic .

The other dyes do not give up to the mark results, when they are added directly to the gel, and it is

required to be stained after electrophoresis is over.

Ethidium bromide is still widely used because it is easy to use, it is economic, and more sensitive tha

other dyes. Though its disposal will always be a matter of concern because of its carcinogenic nature. Still if

students and staff are properly instructed for its handling it will not be a matter or concern.

Loading Dye

Loading dyes which are used in gel electrophoresis have three main roles . They add weight to the

sample, so it gets settled down in the well properly. They provide color and are visible so its easy to load the

sample in the well. The dyes move at steady rate in the gel, so we can get estimation about how far DNA

fragments have move in the gel.

Ficoll & Orange G (6x)(10ml)

1.5g Ficoll 400

Orange G dye

Double distilled water

The dye is stored in small aliquots at 4°C in a regrigerator.

Sucrose & xylene cyanol / bromophenol blue (6x)(10ml)

Sucrose4g

25mg bromophenol blue or xylene cyanol


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This dye is stored at 4°C to avoid any contamination.

Glycerol & bromophenol blue (6x)(10ml)

3ml glycerol (30%)

25mg bromophenol blue (0.25%)


3.7 Applications:

No work of molecular biology is possible without agarose gel electrophoresis

DNA EXPERIMENTS

To visualize Products of animal, plant or bacterial DNA Extractions

To study RFLP

FORENSIC SCIENCE

Paternity test

DNA Fingerprinting

HUMAN HEALTH & DISEASE

To study Human Genomics & Bioinformatics

Restriction Enzyme Mapping

Diagnostics(Human and plant pathogens)

DNA Profiles of ‘Infectious Diseases’

TRANSFORMATION & BACTERIAL CLONING

To screen cloned products

POLYMERASE CHAIN REACTION

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To check amplified DNA

4. Sodium dodecyl sulphate polyacryl amide gel electrophoresis ( SDS PAGE)

Polyacrylamide:

Fig.1 Formation of polyacrylamide

Polyacrylamide is formed by is a linking of acrylamide molecules

The concentration of acrylamide is used between 3.5 and 20%.

Acrylamide is a neurotoxin and needs careful handling

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Polyacrylamide is non-toxic, but polyacrylamide gels should not be touched bare handed because of

there is still possibility that acrylamide is present in free state.

Polyacrylamide gels shows narrow separation range, but their power of resolving two close sizes of

proteins very high

If we want to separate DNA fragments less than 500 bp, polyacrylamide gel is used

DNA fragments differing in length by even a single base pair can also be resolved.

The polymerization reaction starts by formation of free radical with ammonium per sulfate (APS) as

which acts as a initiator and N, N, N’, N’-tetramethylene diamine (TEMED) which acts as a catalyst.

SDS (sodium dodecyl sulfate) is a detergent which can dissolve hydrophobic molecules and has a

negative charge attached to it. If a cell is incubated with SDS, the membranes will get dissolved, and

proteins will be denatured by the detergent also all the proteins will imparted with negative charges.

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Fig.2 Role of SDS in PAGE

Fig.3 Cross section of polyacrlamide gel

Role of Ammonium persulphate and TEMED

Ammonium Persulfate (APS) (NH4)2S2O8 is an oxidizing agent which is used in combination with

TEMED to catalyze the polymerization of acrylamide and bisacrylamide

Ammonium persulfate forms free radicals when it is dissolved in water and it initiate

polymerization of acrylamide solutions.

Percentage of gel is made according to the expected molecular weight of protein we want to study

Table.1 Ingredients of SDS PAGE (30 ml)

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Table 2. Sample/Running Buffer

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Gel Making

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Fig.4 Diagrammatic view of gel making process

Fig.5 Diagrammatic view of gel making process Contd.

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Electrophoresis

Fig. 6. Apparatus set up for SDS PAGE

The different pH buffer system and the stacking gel

A buffer is required for electrophoresis. Usually discontinuous buffer is used that means the pH of buffer in the

gel is different than that of pH of buffer in the gel tank. In stacking gel 6.8 pH is used and running gel 8.8pH

buffer is used.

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Fig.7 Different pH levels used during running of SDS PAGE

So you must be wondering that why this different pH for single gel? We all know that glycine can exist in three

different states of charge depending on pH. positivity, neutral or negativity

As soon as electrophoresis starts, the glycine ions which have negative chargein the pH 8.3 electrode buffer are

forced to enter the stacking gel, where the pH is 6.8. Now glycine acts as zwitterionic (neutrally charged) .

Because of this loss of charge they move very slowly in applied electric field.

The Cl- ions (from Tris-HCl) , move much fast in the electric field and they moves faster than the glycine. The

separation of Cl- from the Tris -ion lead to the formation of very small zone with voltage gradient which pulls

the glycine behind it, results in two small separated fronts of migrating ions; the highly mobile Cl- front,

followed by the slower, mostly neutral glycine front.

4.1 Gel Staining

Gel staining: Coomassie blue

Chemical reagents required:

Brilliant Blue R-250 (BBR)

Sterile distilled water

Solutions:

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Fixing solution: It is prepared by mixing different ratios of methanol(50), acetic acid(10) and

water(40)

Destaining solution: It is prepared by mixing different ratios of methano(45)l, acetic acid(10) and

water(45).

Coomassie concentrated stain solution: It has 12.0 g BBR to which 300 mL Methanol is added then

acetic acid (60ml) is added. After mixing all components it is stirred properly.

Coomassie Working solution: To this 500 mL Methanol we add 30 ml of Coomassie stain solution

and 400ml of water plus 100 ml of acetic acid. After mixing it is filter sterilized using a syringe

filter

Silver Staining

The stain is more sensitive and able to detect protein concentrations from 1 ng to 1 mg.

If gel is not stained properly it can be destained and stained again which is not possible in coomassie

staining.

Silver Staining Protocol

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Table 3. Silver staining Protocol

Destaining Protocol

Destain until no band is visible

Gel is washed 3-5 times for 10 min in a distilled water which should be sterile also till all the stain is

removed

Stain the gel gain using same staining protocol

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4.2 Applications of SDS PAGE

Molecular weight of the proteins can be determined

To perform western blotting SDSPAGE is required

5. 2D-Gel electrophoresis

It is a type of gel electrophoresis where proteins are separated in two dimensions

It is the only method which is available and is capable of simultaneously separating thousands of

proteins.

Basis of separation

Proteins are separated on the basis of two properties.

Firstly they are separated on the basis of their net charge.

After that, PAGE separates the proteins on the basis of their mass.

Using this method small changes in charge and mass can be detected, as it is not possible that two

different proteins will resolve to the same place in both dimensions

Isoelectric Point

There is a pH at which there is no net charge on a protein and this point is called isoelectric point

(pI).

A protein has a net negative charge above its isoelectric point, and it migrates toward the anode in

an electrical field.

The protein is positive below its isoelectric point, and it migrates toward the cathode.

Isoelectric Focusing:

When proteins are separated by isoelectric points it is called isoelectric focusing (IEF).

Therefore, a gradient of pH is applied to a gel and an electric potential is applied across the gel

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proteins will have charge at all pH values other than their isoelectric point,

Proteins move towards the negative end of the gel If they are positively charged and if they are

negatively charged they will move to the positive end of the gel.

In the first dimension proteins will move along the gel and will accumulate at their isoelectric point

5.1 First Dimension Electrophoresis

Commercially available Immobilized pH Gradient (IPG) strips are used for focusing

IPG strips are solid surfaces with coat of dehydrated polyacrylamide

These strips are available in a range of pH gradients such as 5-7, 3-8, 6-12 etc.

Fig.8 IPG Strips

Rehydration of strips is done with rehydration buffer before protein loading

For rehydration of IPG strips these IPG strips are kept in rehydration solution for 10-15 hours.

The focusing is carried out on an equipment which supplies gradient electric current to the IPG strips.

IPG strip has two sides, a base of plastic and the another side which have gel on it.

After this IPG strip is then placed into the lane in a way that the surface with gel faces downwards and

is in direct contact with the solution around it.

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This is left on the working bench for 15 hours.

After 15 hours the IPG strips are removed from the rehydration solution carefully using forceps

This IPG strip is then placed in the fresh tray, with the gel side facing upwards

Fig.9 Isoelectric focusing

Isoelectric Focusing

The strips are put in isoelectric focusing unit

The voltage gradients and time intervals depends on the type of strip that we are using to run the

samples.

The separation can be monitored directly

After focusing the strips are taken out and can be used in next step

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Fig.10. Isoelectric focusing contd

5.2 Second Dimension Electrophoresis

Equilibrium of Strips

We need to equilibrate the strips before separating them in second dimension

It involves the denaturation of proteins, for separation on the basis of molecular weight during SDS-

PAGE.

Equilibration

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Fig11. Second dimension electrophoresis

5.3 Result and Image Analysis

After the proteins gets separated in2DE, each dot on gel is a unique protein which is specific to pI and

molecular weight.

We can not do protein expression profiling manually as the number of spots are very large and manual

interpretation can always lead to false result

We can use commercially available software for analysis

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Fig.12. Gel Image after 2DE

Documents

Gel Electrophoresis, Principle, Types and Applications