WHY TO SEPARATE PROTEINS:
TO STUDY THE STRUCTURE AND FUNCTION OF
TO STUDY THE BASIC PROPERTIES THAT VARY
FROM ONE PROTEIN TO OTHER.
INCLUDING SIZE, CHARGE AND BINDING SITE.
METHODS FOR SEPARATING
Proteins are separated on the basis of their molecular mass ,when placed in an
1st use was reported in 1937 by swedish biochemist ARNE TISELIUS.
He introduced moving boundary electrophoresis - earliest analytical tecnique.
Method takes place in solution.(prevent mixing of migrating proteins)
Requires cumbersome apparatus with large amount of sample.
Zone electrophoresis: sample is forced to move in solid support eg.filter
paper,cellulose acetate or gel.
Largely eleminates convective mixing of samples.
Sample component migrates as discrete zones so, small amount f sample are
the force moving the macro molecule is the electrical potential E , the
electrophoretic mobility of the molecule , (u) is the ratio of the velocity of the
partical ,V to the electric potential.
electrophoretic mobility is also equal to net charge of the molecule ,Z divided
by the frictional coeffiecient f , thus
( u )= v/E = z/f.
Rate of migration depends on:
o Net electrical charge of molecules.
Size & shape of molecule.
Electric field strength.
Properties of supporting medium.
Temperature of operation.
VISUALISED AS WELL AS
DEGREE OF PURITY OF
POINT, MOL.WT, CAN
NOT USED TO PURIFY
PROTEIN IN LARGE
AS IT CAN ADVERSLY
AND FUNCTION OF
TYPES OF ELECTROPHORESIS
a. Agarose gel electrophoresis.
b. Polyacrylamide Gel Electrophoresis
d. Isoelectric Focusing.
e. Two-dimensional Electrophoresis.
Sodium dodecyl sulfate (SDS-PAGE)
Native protein is unfolded by heating
in the presence of -mercaptoethanol
SDS binds to the protein so that it
stays in solution and denatures.
Large polypeptides bind more SDS
than small polypeptides, so proteins
end up with negative charge in
relation to their size.
Thus, we can separate the proteins
based on their mass.
with bound SDS
PROTEIN IS VISUALISED BY ADDING DYE-
COOMASSIE BRILLIANT BLUE.
IT BINDS AND ISOLATES PROTEIN .
PROTEIN BAND FORM IN GEL IN AN DECREASING
MOLECULAR WEIGHT ORDER.
PRESENCE OF UNKNOWN PROTEIN CAN PROVIDE
EXCELLENT MEASURE OF ITS MOL.WT.
Coomassie blue is triphenylmethane dye that binds noncovalently lysyl
residues of proteins. its senstivity is fairly good and it is compatible
with mass spectroscopy.
Figure 6-20 Apparatus for slab gel electrophoresis.
Figure 6-25 Logarithmic relationship between the molecular
mass of a protein and its relative electrophoretic mobility in
A procedure to determine the isoelectric point (pI) of proteins.
thus, a mixture of proteins can be electrophorised through a solution
having a stable pH gradient in from the anode to the cathode and a each
protein will migrate to the position in the pH gradient according to its
isoelectric point. This is called isoelectric focusing.
Ampholytes (amphoteric electrolytes)- low molecular mass (600-900D)
ooligomers with aliphatic amino and carboxylic acid groups with a range of
isoelectric points. Ampholytes help maintain the pH gradiennt in the
presence of high voltage.
Can also use gels with immobilized pH gradients - made of acrylamide
derivatives that are covalently linked to ampholytes. Used with a
gradient maker to ensure continuously varied mixture when the gel is
Figure 6-26 General formula of the ampholytes used
in isoelectric focusing.
pH gradient is established in gel by addition of ampholytes which increases
the pH from anode to cathode.
A protein mixture is placed in a well on the gel.
With an applied electric field ,proteins enter the gel migrates until each
reaches its pH equivalent to its (pI).
Each species of proteins is therby focussed into a narrow band about its pI.
pH 9 -
pH 3 +
General principle and protocol of 2-Dimension Electrophoresis
Traditional Equipment for Isoelectric focusing (IEF):
COMBINATION OF ISO-ELETRIC FOCUSING AND SDS-PAGE
THE GEL IS THEN LAID HORIZONTALLY ON A SECOND GEL AND THE
PROTEINS ARE SEPARATED BY SDS POLYACRYAMIDE GEL
IN THIS TWO-DIMENSIONS GEL ELECTROPHORESIS HORIZONTAL
SEPARATION REFLECTS DIFFRENCES IN pI. VERTICAL SEPARATION
REFLECTS DIFFERENCES IN MOLECULAR WEIGHT..GENARATES ARRAY
OF SPOTS EACH REPRESENTING A PROTEIN UPTO 5000 PROTEINS HAVE
BEEN RESOLVED USING THIS TECNIQUE.
THE INDIVISUAL PROTEIN SPOTS OBTAINED IN STAINED 2 D-GEL IS
REMOVED WITH A SCALPEL,DE-STAINED AND PROTEIN ELUTED FROM
THE GEL FRAGMENTS FOR IDENTIFICATIONS.CHARACTERISATION,BY
MASS SPECTROMETRY.THE 2D ELECTROPHOTGRAM CAN BE ANALYSED
BY COMPUTER AFTER THEY SCANNED & DIGITIZED.
2D gel electrophoresis is generally used as a
component of proteomics.
The step used for the isolation of proteins for further
characterization by mass spectroscopy.
In the lab we use this technique for 2 main purposes:
1.)For the large scale identification of all proteins in a
2.)Differential expression, to compare two or more
samples to find differences in their protein expression.