Chapter 6

Electrophoresis Principle and

Instrumentation
 Introduction
• Electrophoresis is the process of moving charged molecules by applying an
electric field

• Separation of mainly charged materials by differential migration across a

surface or through a column
• The velocity of migration (v) of a molecule is in an electric field is depends on
electric field strength (E), the net charge on the molecule (z) and the frictional
coefficient(f)
V= Ez
f
• The electrical force Ez driving the charge molecule to words oppositely charged
electrode is opposed by viscous drag fv arriving from the friction
• The frictional coefficient f depend on both mass and shape of the migrating
molecule and the viscosity of the medium
Introduction con…
• In general molecules in an electric field move with speed
dependent on
• Their charge
• Shape
• Size
• The molecule will move in the direction of its opposite charge
• Molecule with complex shape and large size will move slowly than
simple molecules
Introduction con…
• Negatively charged molecules (anions) will move toward the
positively charged electrode (the anode)

• Positively charged molecules (cations) will move toward the cathode

(the negatively charged electrode)

• Electrophoresis of macromolecules is normally carried out by

applying a thin layer of a sample to a solution stabilized by a porous
matrix.

• Under the influence of an applied voltage different species of

molecules in the sample move through the matrix at different
velocities.

• At the end of the separation the different species are detected as

bands at different positions in the matrix
Components of the instrument and reagents

1. Power supplies
• The power supply in electrophoretic process is commercially available
electrical power under constant current voltage and power all of
which are adjustable
2. Buffer
• During electrophoresis, current is carried between the cathode and
anode by ions in the buffer solution.
• The function of buffer
– Supply current caring ions
– Maintain desired PH
– Provide a medium for heat dispersion
Supporting medium

• Electrophoresis of macromolecules can be carried out in solution.

However, the ability to separate molecules is compromised by their
diffusion.
• Greater resolution is achieved if electrophoresis is carried out on
semi-solid supports such as polyacrylamide or agarose gels.
• Gels are formed by cross-linking polymers with 99.5% water having
the size of the pores which is similar to the sizes of many proteins and
nucleic acids
• Agarose gels have a larger pore size than acrylamide gels and are
better suited for larger macromolecules.
6
 Support medium

• Type of Support medium

• Starch
• Agarose
• polyacrylamide
Type of matrix

1. Polyacrylamide

Free radicals initiate a vinyl

polymerization reaction

Gels are crosslinked

matrices of acrylamide
polymers
2. Agarose gel
Composed of long unbranched chains of uncharged carbohydrate without cross

links drived from agar

The gels are hydrocolloids, held together by hydrogen and hydrophobic bonds

Large in pore size

Used to separate macromolecules such as nucleic acids large proteins and

protein complexes.
 3. Starch gel

• It was the first material to be used as a support medium for

electrophoresis
• Preparation of reproducible start gel is difficult this medium is now
rarely used in clinical laboratory
Staining reagent
• Stains used to visualize the separated protein fraction
 Gel apparatus

• The chamber contains electrodes which are connected to a power

supply. Thus an electric field is generated across the gel when a
voltage is applied.
• protein has a negative charge and therefore the anode (positive
pole) will be in the lower chamber and the cathode (negative pole)
will be in the upper chamber.

12
General procedure

• The general operation in conventional electrophoresis includes

• Separation
• Staining
• Detection
• Quantification
Types of gel electrophoresis
Type of gel electrophoresis

• Based of gel matrix there are different types of electrophoresis

– Agarose gel for nucleic acid
– SDS PAGE for protein

Importance of gel electrophoresis

• Determine the quality or quantity of DNA
• Estimate the size of DNA molecules
• Purification of DNA
• Analyze PCR products
– Molecular diagnosis or genotyping
1. Agarose Gel electrophoresis

• The technique used to DNA and RNA

• usually used after amplification of DNA via PCR

• After the electrophoresis is complete, the molecules in the gel can

be stained

• If the analyte molecules fluoresce under ultraviolet light,

a photograph can be taken of the gel under ultraviolet lighting
conditions

• The image and the intensity of the band or spot of interest is measured
and compared against standard or markers loaded on the same gel
 Electrophoresis Equipment

Power supply

Cover
Gel tank

Electrical leads


Casting tray

Gel combs
 Chemistry of agarose gel formation

Agarose is a linear polymer of a

galactose-based disaccharide with a
neutral charge

As molten solution of agarose cools, polymers assemble into a gel

32-40 ˚C

85-90 ˚C

Pore size depends on the concentration of agarose

 Preparing the agarose solution
Agar is combined with the appropriate
volume of running buffer in a flask

Flask is microwaved in 20-30 second

bursts, and swirled between bursts

Stop heating when no solid pieces of

agarose powder are visible

Hot solution- beware of steam!

Use a paper towel or other holder
to handle the flask
 Pour the gel

When the flask is cool enough to

handle comfortably, pour the gel

Ends of gel tray should be taped or

placed against a dam

Caution:
Gel apparatus is made of plastic – it can
become deformed if solution is too hot
Form the sample wells

Immediately after pouring the gel, place the sample comb in position

Sample wells form around the teeth of the comb

Allow the gel to thoroughly cure before removing the comb (gel will be
opaque
Prepare the gel for electrophoresis

Remove the dam from the gel (or vice versa)

Orient the gel so that samples will run toward the positive pole

Cover the gel completely with a few mm of running buffer

Agarose gels are sometimes referred

to as “submarine gels”
 Sample Preparation

Mix the samples of DNA with the sample loading buffer (w/ tracking dye).
This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.

Loading Buffer: 
 Bromophenol Blue (for color)
 Glycerol (for weight)
Loading the samples
Samples are mixed with tracking dyes and glycerol
Carefully pipet samples into the wells

Common mistakes:
Bubbles that cause samples to leak from the wells
Puncturing the gel with the pipette tip
Start the electrophoresis
Cover the apparatus with the top
Attach the apparatus to the power supply with the correct polarity
Turn on the power supply, and adjust the voltage (most likely 70-100 volts)
Caution: electrophoresis generates heat
Excessive heat will reduce the resolution of your gel and may even melt it!
Stop the gel when the bromophenol blue is ~2/3 down the gel

xylene cyanol bromophenol blue

Note that condensation increases over time due to the heat

being generated
 DNA molecules are visualized with fluorescent dyes that
intercalate into the DNA helix

Ethidium bromide (EtBr) is a light-sensitive,

water-soluble dye

Fluorescence increases ~20-fold when

bound to DNA

Caution:
Gloves should be worn
when handling EtBr
 Transilluminator and camera system are used to visualize DNA gels

EtBR absorbs ultraviolet light and

emits visible light at ~500 nm

Lift open the door and position

the gel on the transilluminator

Turn on the UV light

AFTER the door is closed
Adjust the exposure and
capture the image
 Lengths of DNA molecules can be estimated from their migration

Restriction digestion of bacteriophage lambda DNA generates a series of

fragments that serve as useful size markers for gels
Factors affect mobility of linear ds DNA?
• Pore size of the gel
–  [agarose]   pore size
–  pore size   friction   mobility
• Voltage across the gel
–  voltage   mobility
• Length of the DNA molecule
– smaller molecules generate less friction and so move faster
Factors affecting resolution

Resolution = separation of fragments

The “higher” the resolution, the more space between fragments of
similar but different lengths.
Resolution is affected by
Agarose concentration
Amount of loaded DNA
Voltage
2. SDS-PAGE (Sodium dodecyl sulfate Polyacrylamide gel electrophoresis)

– Unlike nucleic acids protein can have varying charges and complex

shapes

– Proteins therefore denatured by sodium dodecyl sulfate(SDS) that

coats the proteins with a negative charge.

– The amount of SDS bound is relative to the size of the protein (so that

the resulting denatured proteins have an overall negative charge, and

all the proteins have a similar charge to mass ratio.

– Denatured proteins act like long rods instead of having a complex

tertiary shape, the rate at which the resulting SDS coated proteins

migrate in the gel is relative only to its size and not its charge or shape
SDS-PAGE
• Loading buffer containing sample and

– SDS
• Disrupt
– Hydrogen bond
– Ionic bond
– Hydrophobic bond
– Provide negative charge to the protein when heated at 90

– B-mercaptoethanol

• Cleaved the disulphide bond

– Bromophenol blue
• Used for visualization of the sample on the well

– glycerol
• Increase the density of the sample prevent sample running with buffer
Proteins are prepared by boiling in the presence - -
of SDS
- -

-
-

-
- -

-
-
-

-
- -

-
-

-
-
-

-
-
-

-
Heat -
-
-

-
-
- -
-

-
-
-
- -

-
SDS binding imparts a
-
- - -
constant charge/mass ratio - - -
to the protein -

- -
- - -
- - - -
-

-
Parts of the gel
"Discontinuous" gels have stacking and running gels with different
compositions

Stacking gel
0. 125 M Tris-HCl, pH 6.8 5% acrylamide*
Larger pores, lower ionic strength

Running (resolving) gel

0. 375 M Tris-HCl, pH 8.8 12% acrylamide*

Smaller pores, higher ionic strength

*Investigators adjust the acrylamide

concentration to manipulate the gel pore
sizes

Protein migration varies greatly in the stacking and running gels

Gel preparation
There are two parts of the gel
1. Staking gel (PH 6.8)
– low concentrated polyacrylamide gel (5%) that is placed on the top of
separating gel
– with big pores that do not act as a considerable barrier for large protein
molecules.
– Used to bring all proteins to the separating gel at the same time
– The pH is 6.8. Its pH is acidic implies a lower ionic strength hence a higher
electrical resistance. This provokes the mobility of proteins than other
charged particles present in the gel.
 Parts of the gel..

2. Separating gel (PH 8.8)

– high concentrated polyacrylamide gel depending on protein size (8-
20%) that is placed on the top of separating gel
– with small pores that do act as a considerable barrier for large protein
molecules.
– Used to separate proteins according to the size
Running buffer and protein separation
• Tris-Glycine-chloride (PH 8.3)
– Allowed conduction of current through the gel
– Protein and chloride has negative charge
– Glycine has isoelectric point (PI) of 6 and has neutral charge at PH 6, +ve
charge below PH6 and –ve charge above PH6
– At staking gel(PH 6.8) glycine becomes neutral and move slow after
chloride and protein this helps the protein to be trapped in narrow zone
b/n glycine and chloride
– At separating gel (PH8.8) glycine becomes –ve charge and moves faster
than protein
– Protein will be separated by which higher molecular Wight will move slow
and small molecular Wight will move faster
– Proteins are fixed by acetic acid and stain by coomassie blue
Protein separation
Running buffer: Tris-glycine-SDS, pH 8.3
At the start:

pH 6.8

Protein samples are loaded

into wells
pH 8.8
Bromophenol blue in sample
buffer will act as a tracking
dye
 Protein separation
____
Voltage is applied

Chloride ions in the stacking gel

buffer move rapidly to positive
pole Glycine molecules enter stacking gel

Proteins are negatively charged

and run at various rates
through the stacking gel

Glycine moves more slowly

than the proteins, since very
few glycine molecules have a
negative charge
+
 Protein separation

Differential migration of chloride

and glycine ions sets up a
potential difference that helps
to concentrate proteins

Proteins "stack up" at the

interface between the two gels

pH 8.8

+
 Protein separation

Glycine amino groups lose a

proton as they enter the
running gel

Migration of proteins (invisible) in running

Glycine (now negatively gel is inversely proportional to their
charged) moves more rapidly log(MW)
than proteins at the pH of the
running gel

Proteins are resolved by size

in the running gel

+
Stain by coomassie blue
 Cont’d…
• The amount of dye bound is approximately directly correlated
to the amount of protein.
• The amount of dye bound by each fraction can be simply
determined by cutting out the stained bands and eluting the
dye into a fixed volme of a suitable solvent.
• The absorbance of each solution is measured and the sum of

proteins.

45
 Detection and Quantization of Electropherogram

• Alternative method of quantitation is to

scan the stained electrophoretic strip using
a densitometer,
• The strip is slowly moved across the light
path and the signal from the photoelectric
detector is plotted by a pen recorder, the
area under the trace is proportional to the
total protein content.

46
End of chapter 6