Electrophoresis

By

Muhammad Asif Shaheen

Lecturer,

Department of Pathology,

KEMU Lahore.

Electrophoresis is the migration of charged solutes or particles

in an electrical field.

Iontophoresis refers to the migration of small ions, whereas

zone electrophoresis is the migration of charged

macromolecules in a porous support medium such as paper,

cellulose acetate, or agarose gel film.

An electrophoretogram is the result of zone electrophoresis and

consists of sharply separated zones of a macromolecule. In a

clinical laboratory, the macromolecules of interest are proteins

in serum, urine, cerebrospinal fluid (CSF), and other biologic

body fluids and erythrocytes and tissue.

zone electrophoresis

Any electrophoretic technique in which components are separated

into zones or

bands in a buffer, and stabilized in solid, porous, or any other support

medium–eg, filter paper, agar gel, or polyacrylamidegel

The isoelectric point (pI, pH(I), IEP), is the pH at which a

particular molecule carries no net electrical charge in the statistical

mean. The standard nomenclature to represent the isoelectric point is

pH(I),[1] although pI is also commonly seen

Isoelectric focusing (IEF) is an electrophoresis technique that

separates proteins based on their isoelectric point (pI).

Electrophoresis consists of five components:

The driving force (electrical power),

The support medium,

The buffer,

The sample, and

The detecting system.

Power Supply

Power supplies operating at either constant current or constant

voltage are available commercially. In electrophoresis, heat is

produced when current flows through a medium that has resistance,

resulting in an increase in thermal agitation of the dissolved solute

(ions) and leading to a decrease in resistance and an increase in

current.

The increase leads to increases in heat and evaporation of water from

the buffer, increasing the ionic concentration of the buffer and

subsequent further increases in the current.

The migration rate can be kept constant by using a power supply with

constant current. This is true because, as electrophoresis progresses,

a decrease in resistance as a result of heat produced also decreases

the voltage.

Buffers

Two buffer properties that affect the charge of ampholytes are pH

and ionic strength. The ions carry the applied electric current and

allow the buffer to maintain constant pH during electrophoresis.

An ampholyte is a molecule, such as protein, whose net charge

can be either positive or negative. If the buffer is more acidic than

the isoelectric point (pI) of the ampholyte, it binds H, becomes

positively charged, and migrates toward the cathode. If the buffer

is more basic than the pI, the ampholyte loses H, becomes

negatively charged, and migrates toward the anode.

A particle without a net charge will not migrate, remaining at

the point of application.

Generally, the most widely used buffers are made of

monovalent ions because their ionic strength and molality are

equal.

Support Material

Cellulose Acetate

Cellulose is acetylated to form cellulose acetate by treating it with

acetic anhydride.

Cellulose acetate, a dry, brittle film composed of about 80% air space,

is produced commercially.

When the film is soaked in buffer, the air spaces fill with electrolyte

and the film becomes pliable. After electrophoresis and staining,

cellulose acetate can be made transparent for densitometer

quantitation.

The dried transparent film can be stored for long periods.

Polyacrylamide Gel

Polyacrylamide gel electrophoresis involves separation of

protein on the basis of charge and molecular size. Layers

of gel with different pore sizes are used. The gel is

prepared before electrophoresis in a tube-shaped electrophoresis

cell. The small-pore separation gel is at the

bottom, followed by a large-pore spacer gel and, finally,

another large-pore gel containing the sample. Each layer

of gel is allowed to form a gelatin before the next gel is

poured over it.

Starch Gel

Starch gel electrophoresis separates proteins on the basis of

surface charge and molecular size, as does polyacrylamide

gel. The procedure is not widely used because of technical

difficulty in preparing the gel.

Agarose gel electrophoresis

The term "gel" in this instance refers to the matrix used to

contain, then separate the target molecules. In most cases,

the gel is a crosslinked polymer whose composition and

porosity is chosen based on the specific weight and

composition of the target to be analyzed.

The gel is usually composed of different concentrations

of agarose and a cross-linker, producing different sized mesh

networks. When separating larger nucleic acids (greater than

a few hundred bases), the preferred matrix is purified

agarose. In both cases, the gel forms a solid, yet porous

matrix

"Most agarose gels are made with between 0.7% (good

separation or resolution of large 5–10kb DNA fragments) and

2% (good resolution for small 0.2–1kb fragments) agarose

dissolved in electrophoresis buffer. Up to 3% can be used for

separating very tiny fragments but a vertical polyacrylamide

gel is more appropriate in this case. Low percentage gels are

very weak and may break when you try to lift them. High

percentage gels are often brittle and do not set evenly. 1%

gels are common for many applications

Apparatus for gel electrophoresis

Protocol: Gel Electrophoresis

Pouring a Standard 1% Agarose Gel:

Measure 1 g of agarose.

Note: Agarose gels are commonly used in concentrations of

0.7% to 2% depending on the size of bands needed to be

separated

Mix agarose powder with 100 mL 1xTAE in a microwavable flask.

Microwave for 1-3 min until the agarose is completely dissolved

(but do not over boil the solution, as some of the buffer will

evaporate and thus alter the final percentage of agarose in the

gel. Many people prefer to microwave in pulses, swirling the flask

occasionally as the solution heats up.)

Let agarose solution cool down to about 50°C (about when you

can comfortably keep your hand on the flask), about 5 mins.

(Optional) Add ethidium bromide (EtBr) to a final concentration of

approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock

solution per 100 mL gel). EtBr binds to the DNA and allows you to

visualize the DNA under ultraviolet (UV) light.

Pour the agarose into a gel tray with the well comb in place.

Note: Pour slowly to avoid bubbles which will disrupt the gel. Any

bubbles can be pushed away from the well comb or towards the

sides/edges of the gel with a pipette tip.

Place newly poured gel at 4°C for 10-15 mins OR let sit at room

temperature for 20-30 mins, until it has completely solidified.

Loading Samples and Running an Agarose Gel:

Add loading buffer to each of your digest samples.

Note: Loading buffer serves two purposes: 1) it provides a

visible dye that helps with gel loading and will also allows you

to gauge how far the gel has run while you are running your

gel; and 2) it contains a high percentage of glycerol, so it

increases the density of your DNA sample causing it settle to

the bottom of the gel well, instead of diffusing in the buffer.

Once solidified, place the agarose gel into the gel box

(electrophoresis unit).

Fill gel box with 1xTAE (or TBE) until the gel is covered.

Carefully load a molecular weight ladder into the first lane of the gel.

Carefully load your samples into the additional wells of the gel.

Run the gel at 80-150 V until the dye line is approximately 75-80% of

the way down the gel.

Note: Black is negative, red is positive. (The DNA is negatively

charged and will run towards the positive electrode.) Always Run to

Red.

Turn OFF power, disconnect the electrodes from the power source,

and then carefully remove the gel from the gel box.

(Optional) If you did not add EtBr to the gel and buffer, place the gel

into a container filled with 100 mL of TAE running buffer and 5 μL of

EtBr, place on a rocker for 20-30 mins, replace EtBr solution with

water and destain for 5 mins.

Using any device that has UV light, visualize your DNA fragments

Analyzing Your Gel:

Using the DNA ladder in the first lane as a guide (the

manufacturer's instruction will tell you the size of each band),

you can interpret the bands that you get in your sample lanes

to determine if the resulting DNA bands that you see are as

expected or not.

Cellulose Acetate Electrophoresis

Apparatus

An electrophoresis chamber or tank consists of two compartments

separated by a partition. Each compartment has an electrode

made of an inert material such as platinum. Each side is filled with

equal amount of a suitable buffer solution. A bridge across the top

of the partition holds a membrane or gel with each end of it in

contact with the buffer directly or through paper wicks. The only

connection between the two compartments is through this

membrane.

Sample is applied on to the membrane. Electrical power source is

attached to the tank, which has an indicator for polarity.

Current of prescribed voltage is applied. Molecules start migrating

through the membrane to anode or cathode depending upon their

net charge.

After the prescribed time, current is switched off and the

membrane or gel is removed from the tank.

It is then treated with suitable fixative and is stained to make the

separated bands visible.

Reagents

1. Cellulose acetate strips of suitable size

2. Barbitone buffer, pH 8.6

3. Fixative solution

Ponceau S, 0.5% w/v in 5% trichloracetic acid. Other protein

stains such as commassie brilliant blue (CBB) or amido black

can also be used.

5. Acetic acid, 5% v/v in water as destaining solution.

6. Clearing solution is prepared by adding 15 ml glacial acetic

acid in 85 ml methanol

A typical capillary electrophoresis system consists of a high-

voltage power supply, a sample introduction system, a capillary

tube, a detector and an output device. Some instruments

include a temperature control device to ensure reproducible

results. This is because the separation of the sample depends

on the electrophoretic mobility and the viscosity of the solutions

decreases as the column temperature rises.3 Each side of the

high voltage power supply is connected to an electrode. These

electrodes help to induce an electric field to initiate the

migration of the sample from the anode to the cathode through

the capillary tube.

The capillary is made of fused silica and is sometimes coated

with polyimide.3 Each side of the capillary tube is dipped in a vial

containing the electrode and an electrolytic solution, or aqueous

buffer. Before the sample is introduced to the column, the

capillary must be flushed with the desired buffer solution. There

is usually a small window near the cathodic end of the capillary

which allows UV-VIS light to pass through the analyte and

measure the absorbance. A photomultiplier tube is also

connected at the cathodic end of the capillary, which enables the

construction of a mass spectrum, providing information about the

mass to charge ratio of the ionic species.

Isoelectric focusing

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”Thanks