Lecture Notes - Handouts

10/3/2013

ELECTROPHORESIS

GE - INTRODUCTION
What is Electrophoresis?

Electrophoresis is the study of the movement of
charged molecules in an electric field.

Electrophoresis
is
a
basic
biotechnological
technique/process in which charged biomolecules are
separated according to their mass or charge by the use
of an electric field.
What is Gel Electrophoresis?

Gel electrophoresis involves use of a gel or gelatinous
material which is electrically transparent and acts as a
porous sieve and thus forming a suitable support
medium for electrophoresis of charged molecules.
What are the applications of GE?

GE is used generally for the separation of dyes, proteins
and nucleic acids

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

ELECTROPHORESIS History and Development

1939
1950
1955
1957
1959
1961
1964
1969
1971
1971
1975
1977
1979
1983
1983

S.Raja, A, Dept of Pharmaceutics, IIT (BHU)

3 October 2013

3 October 2013

ELECTROPHORESIS - CLASSIFICATION

Zone electrophoresis developed

Agar gel electrophoresis
Starch gel
Cellulose acetate (Kohn)
Acrylamide gels first used (Raymond and Winstraub)
Isoelectric focusing (IEF)
Disc Gel Electrophoresis (Ornstein and Davis)
SDS electrophoresis (Beber and Osborn)
SDS electrophoresis (Laemmli)
Cellulose acetate gels (Meera)
2-D electrophoresis
Sequencing gels first used
Agarose gel electrophoresis
Pulsed field electrophoresis
Capillary electrophoresis

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

 Capillary Electrophoresis
- FSCE/CZE
- CGE
- CIEF
- CETP
- ACE
- IACE
- NCE

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

 Slab Electrophoresis
- PE
- GE
- SAGE, etc

3 October 2013

Lecture Notes - Handouts

10/3/2013

GE - CLASSIFICATION

CE vs GE

 Agarose Gel Electrophoresis

 Polyacrylamide Gel Electrophoresis
 SDS-PAGE
 Horizontal Electrophoresis
 Vertical Electrophoresis
 Protein Gel Electrophoresis
 Nucleic acid Gel Electrophoresis (DNA GE)



CE - Works according to electrophoretic

Native protein/NA Gel Electrophoresis

Denaturing protein/NA Gel Electrophoresis

 Iso Electric Focusing (IEF)

 Two Dimensional Gel Glectrophoresis (2D GE)
AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

Principle of CE
Samples are introduced into the capillary for separation by two different
methods. Both having advantages and disadvantages; Electrokinetic
injection and Hydrodynamic (Vacuum or Pressure) Injection.

Electrokinetic injection works when the capillary is placed into the

catholyte on one end and into the anolyte (containing the sample to be
analyzed) on the other end. When a voltage is applied, the EOF moves
from the tip of the capillary to the end of the capillary.
A siphoning effect occurs, dragging a representative sample into the
capillary. Also, ions begin moving into the capillary from the buffer
solution due to electrophoretic mobility as part of the sample loading.
This can be an advantage when trying to analyze small concentrations of
these ions. These injections usually last for 1-5 seconds.

GE - Works according to
electrophoretic moment and thus
yielding less resolution than CE.

Have on-line detection thus offering

more optional spectral methods of
analysis of migrating molecules. (Small &
macromolecules)

Have limited applications

proteins and nucleic acids

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

to

GE Theory & Principle

moment and electro-osmotic flow thus

yielding a better resolution than GE

Major Requirements for GE

To perform gel electrophoresis three things are needed.
 Source of current - for the electrical field.
 A buffer system - to maintain constant pH.
 A medium for separation of charged molecules.
(Paper, cellulose acetate, starch, agarose, or polyacrylamide)

Hydrodynamic injection works when a pressure is applied at one end of

the capillary or a vacuum is applied. The pressure differential between
the two opposite sides of the capillary will make liquid move into the
capillary.

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

S.Raja, A, Dept of Pharmaceutics, IIT (BHU)

3 October 2013

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

Lecture Notes - Handouts

10/3/2013

GE Theory & Principle

GE Theory & Principle

 When charged molecules are placed in an electric field, they

migrate toward either the positive or negative pole according
to their charge.
 Proteins which can have either a net positive or net
negative charge move towards respective electrodes
While nucleic acids have a consistent negative charge
imparted by their phosphate backbone, and migrate
toward the anode.
 Proteins and nucleic acids are electrophoresed within a
matrix or "gel".
 The gel is cast in the shape of a thin slab with wells or tube,
and loaded with the sample. The gel is immersed within an
electrophoresis buffer that provides ions to carry a current
and a buffer to maintain the pH.
AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

GE Theory & Principle

 The molecules will be sorted according to their

 Charge
 Size
 Shape
 Small sized molecules with net high charge will move faster and
greater distance in the gel towards the electrodes than large
sized charged molecules.
 A suitable staining dye is used to locate
the position of separated molecules
across the gel.

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

10

GEL ELECTROPHORESIS (GE)

Acrylamide Gel Synthesis

Agarose Gel - Chemistry

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

S.Raja, A, Dept of Pharmaceutics, IIT (BHU)

3 October 2013

11

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

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Lecture Notes - Handouts

10/3/2013

SDS PAGE

GE General Procedure

 Analysis of proteins by gel electrophoresis is not easy

as proteins vary in size and charge
 But in presence of SDS and BME, proteins can be well
easily characterized.
 SDS disrupts the secondary, tertiary and quaternary
structure of the protein to produce a linear
polypeptide chain coated with negatively charged SDS
molecules. 1.4grams of SDS binds per gram of protein.
 Beta-Mercaptoethanol assists the protein denaturation
by reducing all disulfide bonds.

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

13

GE - Instrumentation

S.Raja, A, Dept of Pharmaceutics, IIT (BHU)











Assembling the plates

Prepare gel and buffers
Casting the gels
Prepare samples
Load samples onto gel
Loading the samples
Running the gel
Removing and staining the gel
Interpret and analyze gel

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

14

GE - Instrumentation
Vertical Gel Electrophoresis Assembly

Horizontal Gel Electrophoresis Assembly

AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

General Steps in Gel Electrophoresis

3 October 2013

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AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

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Lecture Notes - Handouts

10/3/2013

GE - Instrumentation

GE General Procedure

Advantages of Vertical Electrophoresis (Esp for proteins)

1) The use of the combination of stacking and resolving gel.
2) Molecular oxygen inhibits polymerization by reacting with the free
radical SO4- ions {Ammonium persulfate (APS) is rather unstable and
decays to produce free radical SO4- ions, which react with the
acrylamide molecules and initiates their polymerization in presence of
Tetramethylethylenediamine (TEMED) catalyze the polymerization of
acrylamide} which is actually the reason why PAGE gels are poured in
between plates and not in open top horizontal apparatuses, as can
be done with agarose.
3) Another advantage with the vertical set-up is that it allows for a much
smaller gel volume, which is useful when making a denaturing gel
which includes several toxic and expensive ingredients than a simple
agarose gel for DNA.
AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

S.Raja, A, Dept of Pharmaceutics, IIT (BHU)

3 October 2013

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AS Raja, Department of Pharmaceutics, IIT (BHU), Varanasi

3 October 2013

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