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ELECTROPHORETIC METHODS

Basic principles of electrophoresis


1. It is the process of moving charged
biomolecules in solution by applying
an electrical field across the mixture.
2. Biomolecules moved with a speed
dependent on their charge, shape,
and size and separation occures on
the basis of molecular size.

Electrophoresis is used: for analysis and


purification of very large molecules
(proteins, nucleic acids)
for analysis of simpler charged
molecules (sugars, amino acids,
peptides, nucleotides, and simpler
ions).
When charged molecules are
placed in an electric field,
they migrate toward either
the positive (anode) or
negative (cathode) pole
according to their charge.

1. Factors influenced
electrophoresis mobility:
2. net charge of the
molecule
3. size and shape
4. concentration of the
molecule in solution
Amino acids have characteristic titration curves

Proton Proton
donor acceptor

At the midpoint – pK=9.60 there


is equimolar concentration of
+ proton donor and proton
acceptor.

Dipolar ion Izoelectric point

At the midpoint – pK1=2.34 there


+ is equimolar concentration of
proton donor and proton acceptor.

Proton Proton
Fully protonated donor acceptor
form at wery low pH
Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Electrophoresis is carried out by applying a thin
layer

Aqueous protein solution is immobilized in a solid hydrophilic


support.

Solid matrix with pores which are used:


• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose

Molecules in the sample move through porous matrix at


different velocity.
• Electrophoresis can be one
dimensional (i.e. one plane of
separation) or two dimensional.

• One dimensional electrophoresis is


used for most routine protein and
nucleic acid separations. Two
dimensional separation of proteins is
used for finger printing , and when
properly constructed can be
extremely accurate in resolving all of
the proteins present within a cell
(greater than 1,500).

• Most common stabilizing media are


polyacrylamide or agarose gels.
Buffers
• Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
• High ionic strength of buffer
– produce sharper band
– produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
Zone electrophoresis

• Much simple method


• Much greater resolution
• Require small sample
• Acetate cellulose – support medium

Protein separation depends on :


• Type and number of ionizable side chains of amino
acids - R.
• Size of net charge (positive or negative).

• Negatively charged proteins move towards the anode.


• Positively charged proteins move towards the
cathode.
Stripe of cellulose acetate

Electrophoresis

Major protein components


separate into discrete zones

Densitometer tracing
density of zones is proportional
to the amount of protein
Example of application of zone electrophoresis in
clinical practice

Hypergamaglobulinemia

Hypogamaglobulinemia

Normal serum
Gel electrophoresis

• Gel is a colloid in a solid form (99% is water).

• Gel material acts as a "molecular sieve.

• During electrophoresis, macromolecules are forced to


move through the pores when the electrical current is
applied.
Support media

• Agarose and polyacrylamide gels are across-


linked, spongelike structure
• It is important that the support media is
electrically neutral. Presence of charge group
may cause:
-Migration retardation
-The flow of water toward one or the other electrode so
called ‘Electroendosmosis (EEO)’, which decrease
resolution of the separation
Agarose – highly purified
polysaccharide derived from
agar (extracted from
seeweed), long sugar
polymers held together by
hydrogen and hydrophobic
bonds.

Acrylamide (CH2=CH-CO-
NH2)
Polyacrylamide gel structure
held together by covalent
cross-links
Agarose gels

• For the separation of (1) large protein or protein


complex (2) polynucleotide 50-30,000 base-pairs
• The pore size is determined by adjusting the
concentration of agarose in a gel (normally in the rank
of 0.4-4%

OH
CH2OHO O

O O
O OH
OH
O
Polyacrylamide gels

CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide

Free radical catalyst


-CH2-CH-CH2-CH-CH2-CH-
CO CO CO
NH NH2 NH
n
CH2 CH2
NH NH2 NH
CO CO CO
-CH2-CH-CH2-CH-CH2-CH-
n
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
• SDS (also called lauryl sulfate) - anionic detergent
• Molecules in solution with SDS have a net negative charge within a wide pH
range.
• A polypeptide chain binds amounts of SDS in proportion to its relative
molecular mass.
• The negative charges on SDS destroy most of the complex structure of
proteins, and are strongly attracted toward an anode (positively-charged
electrode) in an electric field.
Diagrams of vertical slab gel assembly
Determination of molecular mass
Commonly used protein stains

Stain Detection limit

Ponceau S 1-2 mg
Amido Black 1-2 mg
Coomassie Blue 1.5 mg
India Ink 100 ng
Silver stain 10 ng
Colloidal gold 3 ng
Staining with Coomasie blue

1 2 3

Assesment of individual lines


2

3
An ethidium-stained gel photographed under UV light

**Each band that you see is a collection of millions of


DNA molecules, all of the same length!!
Western blott technique

• Western blot (also called immunoblot)


is a technique to detect specifically one
protein in a mixture of large number of
proteins and to obtain information about
the size and relative amounts of the
protein present in different samples.

• In first proteins are separated using


SDS-polyacrylamide gel electrophoresis.

• Then they are moved onto a


nitrocellulose membrane. The proteins
retain the same pattern of separation
they had on the gel.
• An antibody is then added to the
solution which is able to bind to its specific
protein and forms an antibody-protein
complex with the protein of interest. (In
fact there is no room on the membrane for
the antibody to attach other than on the
binding sites of the specific target protein).

• Finally the nitrocellulose membrane is


incubated with a secondary antibody,
which is an antibody-enzyme conjugate
that is directed against the primary
antibody.

• The location of the antibody is revealed


by incubating it with a substrate that the
attached enzyme converts to a product
that can be seen and followed and then
photographed.
Isoelectric focusation

Proteins are separated in pH gradient.


Protein migrate into the point where its net charge is zero –
isoelectric pH.
Protein is positively charged in solutions at pH values below its pI.
Protein is negatively charged in solution at pH above its pI.
Two-dimensional gel electrophoresis
(2-D electrophoresis )
 In the first dimension, proteins are resolved in according to their isoelectric
points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric
focusing (IEF), or non-equilibrium pH gradient electrophoresis.
 In the second dimension, proteins are separated according to their
approximate molecular weight using sodium dodecyl sulfate poly-acrylamide-
electrophoresis (SDS-PAGE).
Electrophoreogram of the mixture of proteins

Protein „maps“ are compare with control pattern of normal


healthy person and abnormalities are analysed
Capillary electrophoresis

Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in


length.
Due to electroosmotic flow, all sample components migrate towards
the negative electrode.
The capillary can also be filled with a gel, which eliminates the
electroosmotic flow. Separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution, greater
sensitivity, and on-line detection.
Electroosmotic flow

The surface of the silicate glass capillary contains negatively-charged


functional groups that attract positively-charged counterions. The
positively-charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. This overall solvent
movement is called electroosmotic flow. During a separation, uncharged
molecules move at the same velocity as the electroosmotic flow (with
very little separation). Positively-charged ions move faster and
negatively-charged ions move slower.