Electroph

oresis

Electropho
resis
Content :
# Background & Theory

# Agarose Gel
Electrophoresis
# Polyacrylamide Gel
Electrophoresis
# IEF & 2-Dimensional
Electrophoresis

Electropho
resis
Principle: Under the influence of an
electrical field, charged molecules
and particles migrate in the
direction of the electrode bearing
the opposite charge.
# The substances are in aqueous
solution

Electrophoresis



Electron =

Phore =



m
(electrophoretic mobility)
(steady-state) m cm2/volt-sec

(Q) (r) (h)

m = Q/Krh

k = constant

(migration velocity;v)

v = EQ/krh

The factors affecting

Electrophoretic separations
1.The sample itself
Charges, size, shape

2. The electric field

Volt, electric current &
heat
3. The medium in which electrophore
Stabilizing medium
Buffer ingredients & concentrati
Electro-osmosis, EO

Apparatus and materials for electrophoresis

1. The electrophoresis chamber

2. Power supplies

3. Supporting medium

4. Buffer

Apparatus and materials

for electrophoresis

1.Moving-boundary
Electrophoresis
2.Paper Electrophoresis

3.Cellulose acetate
electrophoresis
4.Starch Gel
electrophoresis

Moving-boundary
Electrophoresis

Schlieren

Paper Electrophoresis
- Supporting medium : Paper
-

Cellulose acetate electrophoresis

-

- Chemically inert,
-

routine clinical analysis

electrophoresis
Purpose: Screen for hemoglobin variants
Principle: Hb has a net negative charge at pH8.6 and
move in an electrical filed towards the anode (positive)
due to the variation in the amino acid content off
different Hb, the net charge of each Hb types varies
and this will determine their rate of mobility.
Cellulose acetate > easily available
> provides a sharp resolution in a
short time
> allow cleaning,
densitometric quantitiation
> permanent storage of the
transparent film
Procedures:
Electrophoresis the plate for 25 minutes at 350 volts

Cellulose acetate

alkaline electrophoresis
+

Hb
electrophoresis.
cellulose
acetate pH 8.4
1. Normal adult
2. HPFH
(heterozygote)
3. Hb S--HPFH
4. Hb C--HPFH
5. Normal
http://first6weeks.blogspot.com/2007/07/hello-everyone-i-was

Gel electrophoresis
# adjustable gel matrix
# Chemically inert & not exhibit
electroosmosis
# Vertical cylindrical gel rods or
plate or horizontal gel slabs are
available
# Divide into 3 supporting
mediums as : Starch gel, Agarose

Starch Gel electrophoresis

-
-

-

- low reproducibility

Electrophoresis in gels
Gel shapes; Three main
arrangements
1. Tubes/Rod

2. Horizontal slabs

3. Vertical slabs

Electrophoresis in gels
Gel sizes ; 4 size groups
Micro : 3-4 cm long
Mini : 7-8 cm
Standard/general : 15-20 cm
Long : up to 100 cm
Thickness

: ~ 1 mm is frequently use in modern system

: down to 0.3-0.5 mm to avoid temp. gradients

Agarose Gel
Electrophoresis
-Polysaccharide from red seaweed
-Pore size depends on concentration
of agarose
-

(> 10 nm)

Agarose Gel Electrophoresis (Cont.)

1. Zone electrophoresis

, LDH, CK

2. Immunoelectrophoresis
-

(

)

...antigen-antibody-antigen-antibody-antigen-antibody...
-
/

- High specificity & sensitivity

- Divide into 3 principles
Counter Immunoelectrophoresis
Zone electrophoresis/Immunodiffusion
Rocket Immunoelectrophoresis

Agarose Gel Electrophoresis

The standard method for

- Separation & Purification of DNA & RNA fra
- DNA restriction fragment analysis

Horizontal submarine gel use for separatio

Nucleic acid has Phosphate group

negative charge
Equal Charge/mass ratio through molecule
Stain gel with
dyes; ethidium
separate
by fluorescent
size
bromide, SYBR green
Visible under UV light

Pulse field gel electrophoresis

Polyacrylamide gel electrophoresis

Chemically inert & particularly mechanically stable
Clear transparent gel, Very little electroosmosis
Polyacrylamide Polymerization
acrylamide acrylamide methylene-bis
acrylamide cross-link

ammonium persulphate catalyst

TEMED

acrylamide;%T cross-linking;%C

%T

Gel composition and the molecular mass range of

proteins with can be separate on a 2.5%C acrylamide
%T (g/100 ml)

Mr(Dalton)

3-5

> 105

5-10

1.5x105-1x104

10-15

105-104

>15

<1.5x104




PAGE



1.Cylindrical gel VS Slab gel

2.Native gel/ non-denaturing gel

electrophoresis VS Denaturing systems
3.continuous buffer system VS
discontinuous buffer system
4. pH

Native gel/ non-denaturing gel electropho

Blue-Native Polyacrylamide Gel

Electrophoresis
Schgger von Jagow

1991
enzymatically active membrane

Denaturing polyacrylamide gel electroph

detergent

Detergent SDS(sodium dodecyl

sulphate), urea

Separation in continuous buffer

system: simple separation

Separation in discontinuous
buffer system
more than one gel mix, buffer system
Most commonly
2 gels : 1
stacking/spacer/top gel
2
separating/running/resolving gel
3 buffers : 2 for each gel
1 for electrode
compartment
Stacking process
stacking gel

ation in discontinuous system

The stacking process & The stacking gel
# Leading ion-sample-trailing ion
# stacking gel

#

leading ion

(stacked)

The resolving or separating gel

# pH trailing ion

 pH
PAGE

3-10

2.5-11

Discontinuous system
stacking
process
Native gel system pH

Continuous system pH

2

- pH

Polyacrylamide gel electrophoresis (PAGE)

http://www.imb-

- proteins are exposed to the ionic detergent SDS

before and during gel electrophoresis
- SDS denatures proteins, causing multimeric
proteins to dissociate into their subunits
- all polypeptide chains are then forced into extended
conformations with similar charge / mass ratios
- SDS treatment eliminates the effect of differences
in shape
chain length = unique determinant of the migration
rate of proteins
- individual polypeptide chains migrate as a negatively
charged SDS-protein complex through the porous
polyacrylamide gel
- speed of migration is proportional to the size of the
proteins
smaller polypeptides running faster than larger

http://www.imb-jena.de/~rake/
Bioinformatics_WEB/proteins_

Isoelectric
focusing
-Takes place in(IEF)
a pH gradient
-Only for amphoteric substances such as
peptides and proteins

-Generated between the cathode and anode

-A solute will migrate to a point where its net
charge is zero
-At the solutes isoelectric point (pI), migration
stops and the sample is focused into a tight zone

-pI = pH at which a dissolve protein has a net

charge of zero & doesnt move in an electric field

http://mcb.berkeley.edu/courses/mcb102/handouts/Doudna/Lecture4.pdf

2-Dimensional
Electrophoresis
3 4 5 6 7 8 9 10
pH

Pathogen

non-pathogen


electrophoresis
1.Visualization of separated materials
Electropherogram, Densitometer

Color stain : Amido Black,Coomassie

Brilliant Blue R-250, Colloidal Gold
(Progold),Ponceau S, Silver stain
Fluorescence and radioactivity as
localization signals

Biological basis

2. Blotting
- The transfer of large molecules on to the surface of an
immobilizing membrane

Choices in electrophoresis
Media
Electrophoretic system
Size
Format

Separation principle
Gels
Target groups
Target molecules
Native
Modified (during pre-treatment)

Choices in electrophoresis (Cont.)

Detection
Principle
Signal generation

Analysis

Result type
Approach

State-of-the art : Capillary

Electrophoresis
# Two buffer reservoirs

# Very small diameter capillary tube

<0.5 metre in length & < 0.1 mm in
diameter
# High voltage power supply (10-60 kV)
# A detection system :
absorbance
detector
CE
conductivity detector

Electroosmotic flow (EO)

CE
silanol
pH

The aqueous buffer

move in CE
because of
Electroosmotic flow

Comp
uter

 CE
1. DNA Nucleic acid
2.
3.
4.

5.
6. Chiral

References

.
.

, 2549
Hawcroft DW. Electrophoresis :
The basics. USA: Oxford
university press Inc., 1997
Westermeier R. Electrophoresis
in practice. 2nd ed. VCH., 1997