1. BY- NEHA.S.SUTAR
MSC PART -1 BIOTECNOLOGY

2. WHY TO SEPARATE PROTEINS:
 TO STUDY THE STRUCTURE AND FUNCTION OF
INDIVIDUAL PROTEIN.
 TO STUDY THE BASIC PROPERTIES THAT VARY
FROM ONE PROTEIN TO OTHER.
 INCLUDING SIZE, CHARGE AND BINDING SITE.

3. METHODS FOR SEPARATING
PROTEINS:
 ELECTROPHORESIS.
ISOELECTRIC FOCUSING.

4. ELECTROPHORESIS
 Proteins are separated on the basis of their molecular mass ,when placed in an
electric field..
 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
required.
PRINCIPLE:
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.

5.  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.

6. ELECTROPHORESIS:
 ADVANTAGES:
 PROTEINS ARE
VISUALISED AS WELL AS
SEPARATED.
 DEGREE OF PURITY OF
A PARTICULAR
PROTEIN, ISOELECTRIC
POINT, MOL.WT, CAN
BE DETERMINED.
 DISADVANTAGES:
 NOT USED TO PURIFY
PROTEIN IN LARGE
AMOUNT.
 AS IT CAN ADVERSLY
AFFECT STRUCTURE
AND FUNCTION OF
PROTEINS.

7.  TYPES OF ELECTROPHORESIS
a. Agarose gel electrophoresis.
b. Polyacrylamide Gel Electrophoresis
c. SDS-PAGE.
d. Isoelectric Focusing.
e. Two-dimensional Electrophoresis.

8. Sodium dodecyl sulfate (SDS-PAGE)
 Native protein is unfolded by heating
in the presence of -mercaptoethanol
and SDS.
 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.
Native protein
Heat
+
Reductant
+
SDS
Denatured protein
with bound SDS
N
C
-
-
-
-
-
-
- -
-
-
-
-
- -
-
-
-

9. AFTER ELECTROPHORESIS:
 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.

10.  Coomassie blue is triphenylmethane dye that binds noncovalently lysyl
residues of proteins. its senstivity is fairly good and it is compatible
with mass spectroscopy.
Coomassie blue

11. Figure 6-20 Apparatus for slab gel electrophoresis.
Page146

12. SDS-PAGE Run

13. Figure 6-24 SDS-PAGE.
Page149

14. Figure 6-25 Logarithmic relationship between the molecular
mass of a protein and its relative electrophoretic mobility in
SDS-PAGE.
Page149

15. Isoelectric focusing:
 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
made.

16. Figure 6-26 General formula of the ampholytes used
in isoelectric focusing.
Page150

17. METHOD:
 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.

18. sample
pH 9 -
pH 3 +
Isoelectric focusing
(1st dimension)
General principle and protocol of 2-Dimension Electrophoresis
MW
pH
gradient
SDS-PAGE
Ampholytes
polyacrylamide
2nd dimension

19. IEF PRINCIPLE:

20. Traditional Equipment for Isoelectric focusing (IEF):
Ampholytes
polyacrylamide
Cathode
(-)
electrode
solution
Anode (+)
electrode
solution

21. 2-D ELECTROPHORESIS:
 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
ELECTROPHORESIS.
 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.

22. 2 –D ELECTROPHORESIS

23. Figure 6-27 Two-dimensional (2D) gel electrophoresis.
Page150

24. Proteomics solution
IEF
SDS-PAGE

25. 2D Gel Analysis Software

26. Computerized imaging

27.  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
sample.
2.)Differential expression, to compare two or more
samples to find differences in their protein expression.
Applications

28. THANK YOU