This document discusses methods for separating proteins, focusing on electrophoresis techniques. It explains that proteins can be separated based on their size, charge, and binding properties to study each protein's individual structure and function. Electrophoresis methods separate proteins in an electric field based on factors like their molecular mass and charge. SDS-PAGE is described as denaturing proteins with SDS to give each one a uniform negative charge proportional to its size. Isoelectric focusing separates proteins based on their isoelectric point by using a pH gradient gel. Two-dimensional electrophoresis combines these methods to separate thousands of proteins based on both their isoelectric point and size.

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.
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12. SDS-PAGE Run

13. Figure 6-24 SDS-PAGE.
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14. Figure 6-25 Logarithmic relationship between the molecular
mass of a protein and its relative electrophoretic mobility in
SDS-PAGE.
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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.
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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.
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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