This document provides an overview of electrophoresis techniques. It discusses different types of electrophoresis including gel electrophoresis techniques like agarose gel electrophoresis and SDS-PAGE. It explains how agarose and polyacrylamide gels are prepared and the buffers used. It also covers applications of electrophoresis like analyzing DNA, RNA, and proteins, and describes techniques like native PAGE and gradient gel electrophoresis.

1. Electrophoresis
Supervisor
Dr. Moosavi-Nasab
Provisioner
Nazila gharibi
Autumn 1400
1

2.  Electrophoresis
 Types of electrophoresis
 Gel electrophoresis
 Agarose Gel electrophoresis
 SDS-PAGE
 Standard carve
 Native-Page
 Gradient gel
Overview
2

3.  Electrophoresis is the migration of charged particles
or molecules in a medium under the influence of an
applied electric field.
3

4. 4

5. Types of Electrophoresis
Zone electrophoresis
• Gel electrophoresis
• Paper electrophoresis
• Thin layer electrophoresis
• Cellulose acetate electrophoresis
Moving Boundary electrophoresis
• Capillary electrophoresis
• Isotachophoresis
• Isoelectric focusing (IEF)
• Immunochemical
5

6. Types of Gel electrophoresis systems
Horizontal system
 Agarose Gel electrophoresis
(pores: 100 to 500 nm)
DNA, RNA
Vertical system
 Polyacrylamide Gel electrophoresis (PAGE)
(pores: 10 to 200 nm )
Protein
• SDS-Page
• Native-Page
6

7. DNA , plasmid
cDNA , PCR Product
RNA
Extravtion from Gel
Shorter molecules
7

8. Agarose Gel electrophoresis Buffer
 Tris-Borate Tris is a strong base and borate is an acid, combination of both
maintains the pH nearly neutral range of 8 to 8.5. Under this alkaline condition, DNA
is protected and can separate properly.
 EDTA EDTA has some important role to play in this combination. EDTA is a
chelating agent. It chelates the Mg2+ ion which is required for enzyme DNAse as a
cofactor. So by addition of EDTA, our DNA is protected from the enzymatic activity.
Further, the buffer will neutralize the charge of a water molecule.
 TBE Buffer: Tris- Borate-EDTA Buffer PH: 8
 TAE Buffer: Tris- Acetate-EDTA Buffer PH: 8
8

9.  The conductivity of TAE buffer is better so dsDNA can migrate faster as compared to TBE
buffer
 DNA can be easily recovered in TAE buffer so the recovery rate of TAE buffer is higher. It
is a cost-effective and cheaper than other buffer systems
 TAE buffer can interfere with enzymatic reaction and protect DNA, in contrast, TBE buffer
can not.
TBE / TAE buffer in Agarose gel
higher buffering capacity
resolution is very good for longer DNA fragments
9

10. 10

11. Mount
Material
10.8 gr
Tris-base (or HCL)
5.5 gr
Boric acid
./74
./5 M EDTA (PH:8)
10x TBE Buffer ( 100 ml )
50 ml 50 ml
Cassette Buffer tank Buffer ( 500 cc 1xTBE Buffer)
( 20-30 cc 1xTBE Buffer+ Agarose )
(2ml 10xTBE Buffer + 18 ml DW) (50 ml 10xTBE Buffer+ 450ml Dw)
11

12. Final 1x TAE
Buffer volume
Dl water
10X TAE Buffer
concentrate
500 ml
450 ml
50 ml
1000 ml
900 ml
100 ml
1500 ml
1350 ml
150 ml
2000 ml
1800 ml
200 ml
12

13. 𝟐
𝟑
- electrode
13

14. 14

15. 15

16. Applications
 PCR product analysis
 To determine the presence or amount
of DNA and size of DNA fragments
 Gel extraction and DNA recovery
 Plasmid characterisation
 Southern/Northern blotting
 DNA Fingerprinting
 RFLP analysis
 Detection of DNA polymorphisms
 To analyze Restriction digestion
products
• Glycoprotein Electrophoresis
16

17. SDS-PAGE
17

18. SDS-PAGE
SDS-PAGE is an Sodium dodecyl sulphate (SDS) -
polyacrylamide gel electrophoresis (PAGE) method that
most widely used method for:
 analyzing protein mixture qualitatively.
 Useful for monitoring protein purification – as
separation of protein is based on the size of the
particle.
 Can also be used for determining the relative molecular
mass of a protein.
Ulrich K. Laemmli
Unfolding of a protein with SDS
18

19. SDS-PAGE
Stacking gel: ordering/arranging and conc the
macromolecule before entering the field of
separation. (4% of acrylamide)
• Purpose is to concentrate protein sample in
sharp band before enters main separating gel.
Running gel (separating or resolving gel): the
actual zone of separation of the particle /
molecules based on their mobility. (15% of
acrylamide)
Pore size: routinely used as 3% to 30% which is
of pore size 0.2nm to 0.5nm resp.
Reagent : Acrylamide/ Bisacrylamide + Tris-HCL
(PH=8) + 10% SDS + 10% APS+ TEMED
19

20. 10% separation gel ( Down Gel ) 15ml 20ml 30ml
H2o 5.9 7.9 11.9
30% stock 5 6.7 10
1.5 M Tris ( pH:8.8) 3.8 5 7.5
10% SDS 0.15 0.2 0.3
10% APS 0.15 0.2 0.3
TEMED 0.006 0.008 0.012
12% separation gel ( Down Gel ) 15ml 20ml 30ml
H2o 4.9 6.6 9.9
30% acrylamide stock 6 8 12
1.5 M Tris ( pH:8.8) 3.8 5 7.5
10% SDS 0.15 0.2 0.3
10% APS 0.15 0.2 0.3
TEMED 0.006 0.008 0.012
5% stacking gel ( up Gel ) 6ml 8ml 10ml
H2o 4.1 5.5 6.8
30% acrylamide stock 1 1.3 1.6
1 M Tris ( pH:6.8) .75 1 1.25
10% SDS 0.06 0.08 0.1
10% APS 0.06 0.08 0.1
TEMED 0.006 0.008 0.01
Gel preparation ( protocol 1 )
20

21. Gel preparation ( protocol 2)
Separation gel
‫الزم‬ ‫مواد‬
‫ژل‬ ‫درصد‬
6%
7.5%
10%
12%
15%
‫مقطر‬ ‫آب‬
(
ml
)
2.5
9.4
1.4
4.3
4.2
‫تریس‬ ‫بافر‬
5.1
‫موالر‬
pH8/8
6.2
5.2
5.2
5.2
5.2
SDS10%(ml)
1.0
1.0
1.0
1.0
1.0
‫آمید‬ ‫اکریل‬
/
‫امید‬ ‫اکریل‬ ‫بیس‬
(
ml
)
2
5.2
3.3
4
5
TEMED(µl)
5
5
5
5
5
‫پرسولفات‬ ‫آمونیوم‬
10% (ml)
50
50
50
50
50
Stacking gel
‫الزم‬ ‫مواد‬
‫ژل‬ ‫درصد‬
3%
4%
5%
‫مقطر‬ ‫آب‬
(
ml
)
3
3
6.75
‫تریس‬ ‫بافر‬
pH8/8
(
ml
)
1.25
(
1
‫موالر‬
)
1.25
(
5.0
‫موالر‬
)
3
(
0.5
‫موالر‬
)
SDS 10 %
(
µl
)
50
50
120
‫آمید‬ ‫اکریل‬
/
‫امید‬ ‫اکریل‬ ‫بیس‬
(
ml
)
0.625
0.625
2
TEMED(µl)
5
5
5
‫پرسولفات‬ ‫آمونیوم‬
10%
(
µl
)
50
50
50 21

22. Unfolding of a protein with heat
SDS
SDS acts as a surfactant, About 1.4
grams of SDS bind to a gram of protein,
corresponding to one SDS molecule
per two amino acids.
SDS nonpolar chains arrange
themselves on proteins and destroy
secondary tertiary and quarternary
structrure
So much SDS binds to proteins that the
negative charge on the SDS drowns out
any net charge on protein side chains
In the presence of SDS all proteins
have uniform shape and charge per
unit length
Unfolding of a protein with SDS
22

23. Polymerization of Acrylamide
• polyacrylamide gels are formed
from the polymerisation of
acrylamide monomer in the
presence of smaller amounts of
N, N’methylenebisacrylamide
(bis -acrylamide)
• Bisacrylamide polymerizes along
with acrylamide forming cross-
links between acrylamide chains.
 Pore size in gels can be varied by
varying the ratio of acrylamide
to bis-acrylamide.
 Protein separations typically use
a 29: 1 or 37. 5: 1 acrylamide to
bis ratio
23

24. Polymerization of Acrylamide
%T is the total percent of monomer,
MAcr is the mass of acrylamide,
MBis is the mass of bis-acrylamide,
and VSol is the total volume of
solution.
percent total acrylamide (%T) in a gel,
relative percentage and type of
crosslinker (%C)
24

25. 25

26. APS – TEMED(Catalyst of polymerization)
 Polymerization of acrylamide is initiated by the addition of ammonium persulphate
(APS) and the base N, N, N’-tetrametyhlenediamine (TEMED) .
 TEMED catalyses the decomposition of the persulphate ion to give a free radical.
 APS acts as a free-radical initiator, while TEMED catalyzes the polymerization.
26

27. Sample Buffer
 Tris buffer to provide appropriate
pH
 SDS detergent to dissolve proteins
and give them a negative charge
 Glycerol to make samples sink into
wells
 Bromophenol Blue dye to visualize
samples
 Heat to 95 C for 4 minutes
‫ترکیب‬
‫مقدار‬
(
‫میلی‬
‫لیتر‬
)
‫غلظت‬
‫کننده‬ ‫متراکم‬ ‫ژل‬ ‫بافر‬
2
‫سولفات‬ ‫دودسیل‬ ‫سدیم‬
2/3
10
%
‫گلیسرول‬
6/1
‫بتامرکاپتواتانول‬
8/0
‫بلو‬ ‫بروموفنول‬
4/0
1/0
%
‫نهایی‬ ‫حجم‬
8
Sample Buffer 2X
27

28. Staining Proteins in Gels
Chemical stains detect proteins based on differential binding of the stain by the
protein molecules and the gel matrix. They are nonspecific in action, detecting
proteins without regard to their individual identities.
Coomassie Brilliant Blue
 The CBB staining can detect about
1 µg of protein in a normal band.
Silver Staining
 The silver stain system are about
100 times more sensitive,
detecting about 10 ng of the
protein.
28

29. Standard carve
A standard curve, also known as a calibration curve or calibration line, is a type
of graph used as a quantitative research technique.
29

30. Application
- Measuring molecular weight estimation
- Peptide mapping.
- Estimation of protein size.
- Determination of protein subunits
- Estimation of protein purity.
- Protein quantitation.
- Monitoring protein integrity.
- Comparison of the polypeptide composition of
different samples.
- Western blotting.
30

31. 31

32. Western blot
32

33. 33

34.  Sometime, we need to separate protein in non- denaturing conditions. This type
of polyacrylamide gel electrophoresis is also called native gel electrophoresis
because protein remains in native form even after electrophoresis
 The basic difference in the native gel electrophoresis (native-PAGE) is the
electrophoresis buffer does not contain SDS, Also, loading buffer does not have
SDS and reducing agents and samples are not boiled
 Rest of the things are similar to SDS- PAGE gel electrophoresis
Native Page
34

35. Type of Native Page
I. Blue native PAGE
 BN-PAGE is a native PAGE technique, where the Coomassie brilliant blue dye provides
the necessary charges to the protein complexes for the separation without dissociating
them.
II. Clear native PAGE
 CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and
membrane proteins in a polyacrylamide gradient gel. It uses no charged dye so the
electrophoretic mobility of proteins in CN-PAGE is related to the intrinsic charge of
the proteins.
III. Quantitative native PAGE
 The folded protein complexes of interest separate cleanly and predictably due to the
specific properties of the polyacrylamide gel.
35

36. 36

37. protocol
37

38. 38

39. Gradient gel
• This is an polyacrylamide gel system.
• Instead of running a slab of uniform pore size, a gradient gel is formed.
• Uniformly from 5% to 25% acrylamide from top to bottom.
• The highest conc gradient is layed first and than decreasing gradient is poured. But
the sample move down, were the pore size reduces along the path.
39

40. Advantage :
 Greater range of protein can be separated (Complex mixtures can be run).
 You Can Better Separate Similar-Sized Proteins
 Gradients Produce Sharper Bands
40

41. 41

42. 42

43. 43

44. Thank You
44