2. Electrophoresis is a technique whereby charged molecules are
separated by electric field. The charged molecules migrate at
different velocities and are separated.
Usually carried out in aquas solution.
A technique being used in many scientific fields……commonly
used for protein and DNA analyses in Biological sciences
4. This electrokinetic phenomenon was observed for the first time in
1807 by Ferdinand Frederic Reuss (Moscow State University), who
noticed that the application of a constant electric field caused clay
particles dispersed in water to migrate.
History of Electrophoresis
9. GEL Electrophoresis involves the use of gelatinous
material such as Agarose, Acryl amide, Cellulose acetate,
Starch.
GEL acts as a supporting material for samples
separation, and introduces a sieving action which allows
separation of molecules on the basis of their sizes.
GEL matrix viscosity, density and pore size are the
factors affecting samples migration and separation.
Commonly used to separate DNA and proteins.
10. TYPES OF GEL ELECTROPHORESIS
1. Slab Gel
A. Horizontal
B. Vertical
2. Tube Gel
B. On The Basis of Types of Separation
A. On The Basis of Supporting Media
1. No-Denaturing/native (Separation by size and charge;
charge/mass and shape)
2. Denaturing/Non-Native (separation by size)
3. Other Types (IEF, 2-D-GEL)
11. 1. Non-Denaturing/Native (separation by size AND CHARGE)
B. On The Basis of Types Separation
In native PAGE, samples are separated according to the net charge, size and shape of
their native structure.
Electrophoretic migration occurs because most proteins carry a net negative charge in
alkaline running buffers. The higher the negative charge density (more charges per
molecule mass), the faster a protein will migrate. At the same time, the frictional force
of the gel matrix creates a sieving effect, retarding the movement of proteins according
to their size and three-dimensional shape. Small proteins face only a small frictional
force while large proteins face a larger frictional force. Thus native PAGE separates
proteins based upon both their charge and mass.
Because no denaturants are used in native PAGE, subunit interactions within a
multimeric protein are generally retained and information can be gained about the
quaternary structure.
In addition, some proteins retain their enzymatic activity (function) following
separation by native PAGE. Thus, it may be used for preparation of purified, active
proteins.
12. Following electrophoresis, samples can be recovered from a native
gel by passive diffusion or electroelution.
In order to maintain the integrity of samples during electrophoresis, it
is important to keep the apparatus cool and minimize the effects of
denaturation and proteolysis.
Extreme changes in pH should generally be avoided in native PAGE
as they may lead to irreversible damage to protein/DNA of interest,
such as denaturation or aggregation
Native or Non-Denaturing
1. Continuous System: Both gels and electrophoresis tank have same buffer
composition (Single phase gel; a resolving gel)….e.g DNA Agarose gel
electrophoresis
2. Discontinuous System: Both tank and gels have different buffers (two phase
gel; a stacking gel and separating or resolving gel) e.g. Polyacryl amide gel
electrophoresis (PAGE) for proteins
13. 2. Denaturing/Non-Native (separation by size)
A. SDS (Sodium dodecyl sulphate) is used to denature
proteins
(NaC12H25SO4)…. Also called sodium laurilsulfate or
sodium lauryl sulfate
B. Urea or formamide (methaneamide) are used to
denature DNA/RNA
formamide
14. In denaturing PAGE protein samples are heated with SDS before
electrophoresis so that the charge-density of all proteins is made roughly equal.
Heating in SDS, an anionic detergent, denatures proteins in the sample and binds
tightly to the uncoiled molecule.
Usually, a reducing agent such as dithiothreitol (DTT) or
2-mercaptoethanol is also added to cleave protein disulfide bonds and ensure that
no quaternary or tertiary protein structure remains.
Consequently, when these samples are electrophoresed, proteins separate
according to mass alone, with very little effect from compositional differences.
16. 3. Other Types
1. Isoelectric focusing (IEF): Proteins separation based
on isoelectric point in a pH gradient
2. 2-D Gel Electrophoresis: Isolectric focusing followed by
SDS-PAGE
17. TYPES OF GELS
1. Acryl amide
2. Agarose
3. Starch
4. Cellulose Acetate
An agarose is a polysaccharide polymer material,
generally extracted from seaweed. Agarose is a linear
polymer made up of the repeating unit of agarobiose,
which is a disaccharide made up of D-galactose and
3,6-anhydro-L-galactopyranose.
α-(1→3) and β-(1→4) glycosidic bonds
Acryl amide
19. Two Types of Acrylamide gels are used in Electrophoresis
1. Stacking Gel
2. Resolving or Separating Gel
Example recipe for a traditional 10% gel Resolving/Separating for SDS-PAGE.
7.5 mL 40% acrylamide solution
3.9 mL 1% bisacrylamide solution
7.5 mL 1.5 M Tris•HCl, pH 8.8
0.3 mL 10% APS
0.3 mL 10% SDS
0.03 mL TEMED
Add water to 30 mL
Stacking gel has same composition as resolving/separating gel but has the following
two major differences;
1. Stacking gel is always less concentrated than resolving gel
2. pH of stacking gel is approximately 6.8 while resolving gel has pH around 8.8.
3. Stacking gel stacks sample while resolving gel separates them on the basis of their
size, charge and shape or size only.
4. Separting gel is always longer than syctking gel.
20. Electrophoresis can be vertical or horizontal
Stacking and Resolving/Separating Gels
Resolving or Separating gel can be uniform or
gradient
Gradient gel applications include the
determination of protein molecular weights and
the separation of molecules which co-migrate
on uniform gels
21. Protein Molecular Weight Markers
IDENTIFICATION OF SAMPLES
Protein Gel Stains
Coomassie dye
Once protein bands have been separated by polyacrylamide gel electrophoresis, they can
be blotted (transferred) to membrane for analysis by Western blotting (see related article)
or they can be visualized directly in the gel using various staining or detection methods
22. Ponceou S Staining
60 kDa
75 kDa
43 kDa
25 kDa
11 kDa
110 kDa
(50.00g/L acetic acid; 1.0g/L Ponceau S.)
C22H16N4O13S4
672.64 g mol−1
Ponceau S, Acid Red 112, is a sodium
salt of a diazo dye of a light red color,
that may be used to prepare a stain for
rapid reversible detection of protein
bands. A Ponceau S stain is useful
because it does not appear to have a
deleterious effect on the sequencing of
blotted polypeptides and is therefore one
method of choice for locating
polypeptides. It is also easily reversed
with water washes, facilitating
subsequent immunological detection
24. Steps in PAGE for Proteins
1. Preparation of sample
2. Preparation of Gel (s) and Running Buffer (Tank
Buffer; COMPOSED OF Glycine and Tris-HCl)
3. Application of Sample on a Gel
4. Eelectrophoretic migration
5. Staining, Elution, Identification of Sample
All these steps are same in SDS-PAGE (Denaturing) and Native-
PAGE (Non-Denaturing Gel Electrophoresis)
25. Factors Affecting Separation in Gel Electrophoresis
1. Resistance (Pore Size)
2. Buffer Strength
3. Gel Temperature
4. Sample
26. 1. Resistance/Pore Size
Pore size depends on gel percentage
Gel Percentage 1/α Samples Molecular size
The size of the pores created in the gel is inversely related to the amount of
acrylamide used.
A 7% polyacrylamide gel has larger pores than a 12% polyacrylamide gel.
Gels with a low percentage of acrylamide are typically used to resolve large
proteins, and high percentage gels are used to resolve small proteins.
"Gradient gels" are specially prepared to have low percent-acrylamide at the
top (beginning of sample path) and high percent-acrylamide at the bottom
(end), enabling a broader range of protein sizes to be separated.
27.
28. 2. Buffers
Common Buffer components include; Acetic acid, Boric acid,
Glycine, Tris (trisaminomethane; (HOCH2)3CNH2)
Ionic strength of Buffer
pH of Buffer
29. 3. Temperature
Temperature management is a critical to achieve good results
Some applications require high temperature e.g. Denaturing
PAGE of DNA/RNA
Other applications require low temperature to prevent sample
degradation or gel melting
31. Applications of Electrophoresis
1. Identification of Proteins and Nucleic acids
2. Separation and Purification
3. Study of Protein-Protein Interaction (immunoprecipitation)
4. Study of expression level of proteins and nucleic acids
5. Preparation of proteins and nucleic acids for sequencing
6. Study of immunological reactions (Ag-Ab reactions by Wesetern
blotting)
