Electrophoresis

• Submitted by

• Saubhagya

• Sri venkateswara college

• University of Delhi

Electrophoresis Principle & Application

• It is the process of moving charged biomolecules in

solution by applying an electrical field across the mixture.

• This technique was first developed by Arne Tiselius in 1930

for the study of serum proteins.

• Their rate of migration (electrophoretic mobility) through

the electrical field, depends on following factors

• strength of the field,

• net charge, size, and shape of the molecules,

• concentration of the molecule in solution

• ionic strength of buffer, viscosity, and temperature of

the medium in which the molecules are moving.

• Applied voltage

• Electrophoresis is used for:

• For analysis and purification of very large molecules

(proteins, nucleic acids)

• For analysis of simpler charged molecules (sugars,

amino acids, peptides, nucleotides, and simpler ions).

Introduction

• Electrophoresis can be one dimensional (i.e. one plane of

separation) or two dimensional.

• One dimensional electrophoresis is used for most routine

protein and nucleic acid separations. Two dimensional

separation of proteins is used for finger printing , and when

properly constructed can be extremely accurate in resolving

all of the proteins present within a cell (greater than 1,500).

• Most common stabilizing media are polyacrylamide or

agarose gels.

• Molecules can have distinct charges

• Positive or Negative

• Net charge will cause different movement through gel

• Molecules can have different shapes

• Linear

• Globular

• Alpha helix

When charged molecules are placed in an electric field, they

migrate toward either the positive (anode) or negative (cathode)

pole according to their charge.

Basic Principle of Electrophoresis

Electrophoretic mobility

• The principle involved in both slab and capillary electrophoresis are same which involves electrophoreticmobility of the ions.

• Electrophoretic mobility μ = v / E = q / f

– V=Velocity, E=Electric field, q=Charge f=Frictional coefficient

• Depends on

– Particle property :Surface, charge, density & size

– Solution properties: Ionic strength, pH, Conductivity,viscosity

– Temperature & Voltage

General operations performed in conventional

electrophoresis include:

(1) separation

(2) staining

(3) detection

(4) Quantification

General Procedure

The Basic Components

• Support Medium (Powdered of agarose or polyacrylamise

• The electric power supply provides the electricity that carries the molecules through the gel

• The electrophoresis chamber is where it all takes place.

• Boiling Buffer solution• Gel stain

• The “gel” in gel electrophoresis is the component that

physically separates the molecules.

• Use the proper gel concentration for sample size

range.

0.5–5% agarose (for DNA & RNA)

3.5–20% polyacrylamide (for Protein)

• Agarose and polyacrylamide gels are across-linked,

spongelike structure

• It is important that the support media is electrically

neutral. Presence of charge group may cause:

-Migration retardation

-The flow of water toward one or the other electrode so

called ‘Electroendosmosis (EEO)’, which decrease

resolution of the separation

Support media

Agarose – highly purified polysaccharide derived from

agar (extracted from seeweed), long sugar polymers held

together by hydrogen and hydrophobic bonds.

Acrylamide (CH2=CH-CO-NH2) Polyacrylamide gel

structure held together by covalent cross-links

Buffer additives modify sample molecules.

• Commonly used buffer

• Tris-acetate-EDTA , Tris Phosphate EDTA & Tris-borate-

EDTA (50mmol/L; pH 7.5-7.8) used most often for DNA.

• 10 mM sodium phosphate or MOPS buffer used for RNA.

• Barbital buffer & Tris-EDTA for protein

• Function of buffer

1. carries the applied current

2. established the pH

3. determine the electric charge on the solute

• High ionic strength of buffer

– produce sharper band

– produce more heat

Buffers

Combs are used to put wells in the cast gel for sample loading.

– Regular comb: wells separated by an “ear” of gel

– Hound stooth comb: wells immediately adjacent

• Use the proper comb (well) and gel size.

Combs

Applied voltage

• Increase:

-charged molecule migrate faster

-Increases current

- temperature

Temperature

• Better at low temp(40C) : Otherwise density difference convection current Disturbed diffusion

• Distortion of Zones: Column gel migrating Center (warmer) faster than outer (cooler)

• Evaporation: increase in ionic strength of the buffer

• Viscosity: agarose gels becomes softer

Classification

1. Gel electrophoresis

1(a) Electrophoreis of nucleic acids

Agarose gel electrophoresis of DNA

Pulsed- field gel electrophoresis

1(b) Electrophoesis of proteins

Sodium dodecyl sulphate (SDS)- polyacrylamide gel

electrophoresis

– Native (buffer) gels

– Gradient gels

– Isoelectric focusing gel

Two-dimentional polyacrylamide gel electrophoresis

– Cellulose acetate electrophoresis

– Detection estimation and recovery of proteins in gels

– Protein (western blotting) blotting

2. Capillary electrophoresis

3. Microchip electrophoresis

Electrophoresis

• Horizontal Agarose Gels

• Agarose forms a gel or molecular sieve that

supports the movement of small materials in solution

used for DNA

• Vertical Polyacrylamide Gels

• Made of Polyacrylamide

• Used for Protein molecular size, shape, charge

• IEF electrophoresis

• Western Blot technique

Horizontal Gels Vertical gel

• Gel is a colloid in a solid form (99% is water).

• The separation here is brought about through molecular

sieving technique, based on molecular size of substances.

• During electrophoresis, macromolecules are forced to move

through the pores when the electrical current is applied.

Support media

• Agarose for nucleic acid and polyacrylamide gels for protein

are a cross-linked, sponge like structure.

• It is important that the support media is electrically neutral.Presence of charge group may cause:

-Migration retardation

-The flow of water toward one or the other electrode so

called ‘Electroendosmosis (EEO)’, which decreaseresolution of the separation

Gel electrophoresis

Agrose gel electrophoresis

• Agarose gel electrophoresis is a method to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electricfield (electrophoresis).

• The pore size is determined by adjusting the concentration of

agarose in a gel (normally in the rank of 0.4-4%

• Shorter molecules move faster and migrate farther than longer ones .

PFGE allows investigators to separate much larger pieces of DNA than conventional agarose gel electrophoresis. In conventional gels, the current is applied in a single direction (from top to bottom). But in PFGE, the direction of the current is altered at a regular interval

Pulsed Field Gel Electrophoresis (PFGE)

• Detect bands by staining during or after electrophoresis

• Ethidium bromide: for double-stranded DNA

• SyBr green or SyBr gold: for single- or double-stranded DNA or for RNA

• Silver stain: more sensitive for single- or double-stranded DNA or for RNA and proteins

Nucleic acid stain

An ethidium-stained gel photographed under UV light

1(a). Electrophoresis of proteins Sodiumdodecylsulphate(SDS)-Polyacrylamide Gel Electrophoresis• Polyacrylamide gel electrophoresis (PAGE), describes a technique widely

used in biochemistry, forensics, genetics, molecularbiology and biotechnology to separate biological macromolecules, usuallyproteins or nucleic acids, according to their electrophoretic mobility.

• Mobility is a function of the length, conformation and charge of themolecule

• As with all forms of gel electrophoresis, molecules may be run intheir native state, preserving the molecules' higher-order structure, or achemical denaturant may be added to remove this structure and turn themolecule into an unstructured linear chain whose mobility depends onlyon its length and mass-to-charge ratio.

• For nucleic acids, urea is the most commonly used denaturant. For

proteins, sodium dodecyl sulfate (SDS) also called lauryl sulfate is ananionic detergent applied to protein sample to linearize proteins and toimpart a negative charge to linearized proteins. This procedure iscalled SDS-PAGE.

• SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures, and additionally applies a negative charge toeach protein in proportion to its mass.

• Urea breaks the hydrogen bonds between the base pairs of the nucleicacid, causing the constituent strands to separate. Heating the samples toat least 60°C further promotes denaturation.

• In most proteins, the binding of SDS to the polypeptide chain imparts aneven distribution of charge per unit mass, thereby resulting in afractionation by approximate size during electrophoresis.

• Proteins that have a greater hydrophobic content, for instance manymembrane proteins, and those that interact with surfactants in theirnative environment, are intrinsically harder to treat accurately using thismethod, due to the greater variability in the ratio of bound SDS.

• In addition to SDS, proteins may optionally be briefly heated to nearboiling in the presence of a reducing agent, such as dithiothreitol(DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which furtherdenatures the proteins by reducing disulfide linkages, thus overcomingsome forms of tertiary protein folding, and breaking up quaternaryprotein structure (oligomeric subunits). This is known as reducing SDS-PAGE.

SDS -PAGE

Electrophoretic method that separates proteins according

to the iso-electric points

Is ideal for seperation of amphoteric substances

Each protein has own pI = pH at which the protein has

equal amount of positive and negative charges (the net

charge is zero)

PI of proteins can be theoretically predicted. Therefore, IEF

can also be used for protein identification.

Seperation is achieved by applying a potential difference

across a gel that contain a pH gradient

Mixtures of ampholytes, small amphoteric molecules with

high buffering capacity near their pI, are used to generate

the pH gradient.

Isoelectric focusing requires solid support such as

polyacrylamide gel

Isoelectric Focusing

In the first dimension, proteins are resolved in according to their isoelectric points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis.

In the second dimension, proteins are separated according to their approximate molecular weight using sodium dodecyl sulfate poly-acrylamide-electrophoresis (SDS-PAGE).

The combination of these two technique to give two-dimension (2-D)PAGE provides a highly sophisticated analytical method for analysingprotein mixtures.

2D-PAGE

Stain Detection limit

Ponceau S 1-2 mg

Amido Black 1-2 mg

Coomassie Blue 1.5 mg

India Ink 100 ng

Silver stain 10 ng

Colloidal gold 3 ng

Commonly used protein stains

Protein „maps“ are compare with control pattern of normal

healthy person and abnormalities are analysed

Electrophoreogram of the mixture of proteins 2D-gel (coomassie stained)

Example of silver stained gel

Silver staining is usually 10-100 times more sensitive than Coomassie Blue staining, but it is more complicated.

Faint but still visible bands on this gel contain less than 0.5 ng of protein!

Capillary electrophoresis

Capillary gel electrophoresis is used for separation of biological

molecule including amino acid, peptides, proteins, DNA fragments, and

nucleic acids well as any number of small organic molecules such as drugs or even metal ions. to separate nucleic acids.

It is classic techniques of electrophoresis are carried out in a small-

bore, fused silica capillary tube, the outer diameter of such tubes

typically varies from 180 to 375 micrometer, the inner diameter from 20

to 180 micrometer, and the total length from 20 cm up to several

meters.

The capillary can also be filled with a gel, which eliminates the

electroosmotic flow. This capillary tube serves as a capillary

electrophoretic chamber that is connected to a detector at its terminal

end and, via buffer reservoirs, to a high-voltage power supply

Separation is accomplished as in conventional gel electrophoresis but

the capillary allows higher resolution, greater sensitivity, and on-line

detection.

One advantage of using capillary is that they reduce problem resulting from

heating effects. Because of small diameter of the tubing there is a larger

surface-to-volume ratio, which give enhanced heat dissipation. Improved

heat dissipation permits the application of voltages in the range of 20 to 30

kV, which enhances separation efficiency and reduces separation time in

some cases to less than 1 minute

•During a separation, uncharged molecules move at the same velocity as

the electroosmotic flow (with very little separation). Positively-charged

ions move faster and negatively-charged ions move slower.

•The surface of the silicate glass capillary contains negatively-charged

functional groups that attract positively-charged counterions. The

positively-charged ions migrate towards the negative electrode and carry

solvent molecules in the same direction. This overall solvent movement is

called electroosmotic flow.

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3. Microchip electrophoresis

• Microchip analysis completed in tens of seconds where as capillaryelectrophoresis can take 20 min and conventional gel electrophoresis atleast2h.

• Using new detection system, such as laser induced fluorescence,picomole to attomole (10-8 moles) sensitivity can be achieved, which is atleast two order of magnitude greater than for conventional capillaryelectrophoresis.

• The microchip provide an electrophoretic system similar to CE but withmore flexibility.

• Current developments of this technology are based integrating functionsother than just separation in to the chip.

• For example: sample extraction, pre concentration of sample prior toseparation, PCR amplification of DNA sample using infrared mediatedthermo cycling for rapid on-chip amplification and the extraction ofseparated molecules using micro chamber –bound solid phases are allexamples of where further functions have been built into a microchipelectrophoresis system.

• An interface has also been developed for microchip electrophoresis-massspectrometry where drug have been separated by MCE and thenidentified by MS.

Applications

The vast application of electrophoresis include:

• Vaccine analysis such as influenza vaccine, hepatitis vaccine and polio vaccine.

• Protein and DNA analysis

• To see the map and the differences in the genetic code of species on the earth.

• Electrophoretic DNA analysis also provides a reliable tool in forensic investigations.

• Determination of impurities

• Chiral analysis

• Analysis of carbohydrates and other macromolecules.

• Analysis of inorganic anions/metal ions

Protein analysis• Electrophoresis has advanced our understanding on the structure and

function of proteins. These molecules are needed by our body cells andmay be analyzed, for instance by getting blood and urine samples. Thenthrough electrophoresis, the amount of protein in your blood or in yoururine is measure d and compare to established normal value lower orhigher than the normal levels usually indicates a disease.

DNA analysis• Electrophoresis is one way of analyzing DNA, which is the unique code of

every individual. Through electrophoresis, specific DNA sequences can beanalyzed isolated and cloned. The analyzed DNA may be used in forensicinvestigation and paternity tests.

• For this well are formed at one end of an agarose gel for the loading ofthe DNA sample. The slab is then placed horizontally in to theelectrophoresis buffer chamber. The DNA migrates in bands towards thepositive electrode. The smaller molecule migrate through the matrix morerapidly than the larger ones, which are restricted. The DNA bands are thenstained gel is then viewed directly under ultraviolet light andphotographed.