What is electrophoresis

Electro=Electric; phoresis=Migration

Electro refers to electron flow or current.

Phoresis refers to movement.

Thus Electrophoresis is movement under electric current.

This technique therefore can separate molecules which can move in an electric

field i.e charged molecules

Charged molecules in an electric field behave in a predictable manner.

Positively charged molecules will move towards the negative pole while

negatively charged molecules move towards the positive pole.

Definition: The migration of charged particles in solution, proteins and nucleic

acids, under the influence of electrical field.

• Electrophoresis is a method whereby charged molecules in solution, chiefly

proteins and nucleic acids, migrate in response to an electrical field.

• Their rate of migration through the electrical field, depends on the strength of the

field, on the net charge, size, and shape of the molecules, and also on the ionic

strength, viscosity, and temperature of the medium in which the molecules are

moving.

• As an analytical tool, electrophoresis is simple, rapid and highly sensitive.

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 • It can be used analytically to study the properties of a single charged species

or mixtures of molecules.

• It can also be used preparatively as a separating

• Electrophoresis is usually done with gels formed in tubes, slabs, or on a flat

bed.

• In many electrophoresis units, the gel is mounted between two buffer

chambers containing separate electrodes, so that the only electrical

connection between the two chambers is through the gel.

Technique

The rate of migration through the electrical field depends on:

a- The strength of the field

b- The net charge

c- The size and shape of the molecules

d- The ionic strength, viscosity and temperature of the medium in which the

molecules are moving.

Electrophoresis –Separates:

–Nucleic acids –Proteins –Peptides

–Amino acids –Organic acids/bases –Drugs

–Pesticides –Inorganic anions/cations.

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Types of Electrophoresis:

Moving boundary electrophoresis-

In this method, the electrophoresis is carried in solution, without a supporting

media. The sample is dissolved in buffer and molecules move to their respective

counter charge electrodes.

• Moving boundary electrophoresis is carried out in a U shape tube with

platinum electrodes attached to the end of both arms.

• At the respective ends, tube has refractometer to measure the change in

refractive index of the buffer during electrophoresis due to presence of

molecule.

• Sample is loaded in the middle of the U tube and then the apparatus is

connected to the external power supply.

• Charged molecule moves to the opposite electrode as they passes through

the refractometer, change can be measured.

• As the desirable molecule passes, sample can be taken out from the

apparatus along with the buffer.

Disadvantages of Moving Boundary electrophoresis-

The resolution of the technique is very low due to the mixing of the sample as well

as over-lapping of the sample components.

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The electrophoresis technique is not good to separate and analyze the complex

biological sample instead it can be used to study the behavior of the molecule in an

electric field.

Gel Electrophoresis

How Phenomena works

1- The proteins are dissolved in SDS and then electrophorised.

2- Binds to protein in a ratio of one SDS molecule/two amino acids.

3- Masks‘ charge on protein so that all proteins are uniformly negatively charged.

4- The proteins denatured by SDS are applied to one end of a layer of

polyacrylamide gel submerged in a buffer.

5- Buffer provide uniform pH and ions for conducting electric potential.

6- When an electric current is applied across the gel, the negatively-charged

proteins migrate across the gel to the positive pole.

7- Short proteins will more easily fit through the pores in the gel and move fast,

while larger ones will have more difficulty.

8- Due to differential migration based on their size, smaller proteins move farther

down the gel, while larger ones stay closer to the point of origin.

9- After a given period of time, proteins might have separated roughly according to

their sizes.

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11- Proteins of known molecular weight (marker proteins)can be run in a separate

lane in the gel to calibrate the gel.

12- Gel electrophoresis separates molecules on the basis of their charge and size.

13- The charged macromolecules migrate across a span of gel because they are

placed in an electrical field.

14- The gel acts as a sieve to to retard the passage of molecules according to their

size and shape.

The gel is made from agar Agar comes from sea weed.

• DNA is a negative molecules

• Molecules sort based on

• Charge

• Size

• shape

- Use of gelatinous material as a support medium.

- The gel is a matrix that contains pores through which the particles drawn when an

electrical current is applied.

- Used to separate proteins or nucleic acids (DNA, RNA).

Migration Depends on

•Strength of electric fields. •Temperature •Features of the molecule

–Net charge of molecule –Size of molecule –Shape of molecule

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•Features of the Gel

–Gel type –Gel concentration

•Buffer Type/pH.

Gel types:

Starch: Rarely used.

Polyacrylamide: Separation of protein, small nucleic acid fragments.

Agarose: Separation of Nucleic acids, large proteins.

Cellulose acetate: Separation of Proteins.

The most commonly used types are polyacrylamide and agarose.

1) Pulsed Field Gel Electrophoresis

- Based on the periodically changes of directions in the electric field.

- Used for separating very large DNA molecules.

2) Capillary Electrophoresis

- Electrophoresis are carried out in capillary tube (11-111 μm diameter 21 to

211 cm length)

Applications:

Analyzing proteins in physiological matrices (eg. Serum, urine).

DNA analysis.

Drug screening.

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3) Isoelectric Focusing

- Separation of proteins by their isoelectric points (pI) by using pH gradient of the

gel.

2D Electrophoresis

- Separation of proteins by two properties (eg: pI and size) in a mixture.

Components of Gel Electrophoresis System

1) Power Supply Direct current needed to set up an electrical field across the gel.

2) Gel Box It is a container that holds the gel submerged in running buffer*.

*Running Buffer A solution used to carry the electrical current though the gel.

Heat is generated by the application of a current to the gel, the running buffer also

helps keep the gel cool.

46 Wells: Wells are small indentations created in the gel when it is made.

The wells are uniformly spaced along the side of the gel closest to the negative

electrode.

The wells also allow the samples to be placed into the gel so that when current is

applied, the samples are pulled through the middle of the gel, not across the top.

Loading Dye: It is a colored buffer mixed with the material prior to loading onto

the gel. This makes the solution denser than the surrounding running buffer so that

when a sample is pipetted over top of a well it sinks down into the well. Coloring

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the sample provides quick conformation that the samples have sunk into the wells

and makes it easy to keep track of which wells have already been loaded.

Vertical Gel Box

• Different gels thickness can be used.

• More than one gel per apparatus.

• Not easily adapted for different techniques.

• No technician friendly.

• Used for polyacrylamide gel electrophoresis

Horizontal (Flat bed) Gel Box

• Gel thickness limited.

• Only one gel per apparatus.

• Easily adapts different techniques.

• Technician friendly.

• More safe for electricity accidents.

• Used for agarose gel electrophoresis.

Procedure for separation of DNA:

1. Prepare the gel box by adding enough running buffer so that the gel will be

completely submerged once it is place in the gel box.

2. Place the gel in the gel box making sure the gel is completely submerged in the

buffer and that the wells are oriented properly (closest to the negative electrode).

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3. Add loading dye to the sample and standard*.

4. Pipette a small volume of sample/standard into each well.

5. Connect the power supply electrodes to either end of the gel box.

*DNA Standards Due to the many factors that affect the rate of migration of DNA

through the gel, estimating the exact length of a band in the gel must be done

relative to the position of other bands in the same gel.

A standard is placed in one of the wells. By comparing the movement of the

fragments of known length in the standard with the fragments in the sample, an

accurate estimate of the length of the DNA strands in the sample can be made. The

standard is treated the same way as the samples: mixed with loading dye and added

to one of the wells in the gel.

Loading sample onto the gel

Specific Applications:

1) Neoplastic disorders

– Detection of tumor-related mutation.

– Microsatellite instability

– Analysis of monoclonality

2) Diagnosis of hereditary diseases and prenatal testing