1. Electrophoresis Chapter 3-Lecture 7
If a mixture of electrically charged biomolecules is placed in an electric field of field strength E, they will freely move towards the electrode of opposite charge.
Dr Gihan Gawish
Chapter 3-Lecture 7

2. Theory of Electrophoresis
Electrophoretic separations are based upon the fact that the electrical force (F) on a charged particle (ion) in an electrical field (E) is proportional to the charge of the particle (q),
F = qE
The migration of the charged particle in the electric field, called the electrophoretic mobility (μ),
μ = v/E = q/f
Dr Gihan Gawish

3. Theory of Electrophoresis
Move at quite different depending on
physical characteristics of the molecule
experimental system used.
Dr Gihan Gawish

4. Theory of Electrophoresis
Eq = עf
f is the frictional coefficient, describes frictional resistance to mobility and depends on a number of factors such as the mass of the molecule
ע is the velocity
E is the electric field
q is the net charge on molecule
Dr Gihan Gawish

5. 1- Moving Boundary Electrophoresis
Electrophoresis in a free solution.
the separation of colloids through electrophoresis
the motion of charged particles through a stationary liquid under the influence of an electric field.
Developed by Arne Tiselius in Tiselius was awarded the Nobel Prize in chemistry for his work
Dr Gihan Gawish

6. Tiselius Electrophoresis
Moving boundary electrophoresis is electrophoresis in a free solution.
The apparatus includes a U-shaped cell filled with buffer solution and electrodes immersed at its ends.
On applying voltage, the compounds will migrate to the anode or cathode depending on their charges.
The sample applied could be any mixture of charged components like a serum
Dr Gihan Gawish

7. Tiselius Electrophoresis
Moving boundary electrophoresis is electrophoresis in a free solution.
The principle is the motion of charged particles through a stationary liquid under the influence of an electric field.
59% 8% 5%
12%
16%
Dr Gihan Gawish

8. 2- Zone Electrophoresis
separation cellulose acetate electrophoresis.
A drop of sample is applied in a band to a thin sheet of supporting material, like paper, that has been soaked in a slightly-alkaline salt solution.
Plasma protein example: At pH 8.6, which is commonly used, all the proteins are negatively charged, but some more strongly than others.
Dr Gihan Gawish

9. Zone Electrophoresis
A direct current can flow through the paper because of the conductivity of the buffer
The serum proteins move toward the positive electrode.
The stronger the negative charge on a protein, the faster it migrates.
Dr Gihan Gawish

10. Zone Electrophoresis After 20 min, the current is turned off
the proteins stained to make them visible
The separated proteins appear as distinct bands.
serum albumin moves closest to the positive electrode, why?
Dr Gihan Gawish

11. 3- Gel Electrophoresis
The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules.
In most cases the gel is a cross linked polymer whose composition and porosity is chosen based on:
the specific weight
composition of the target to be analyzed.
Dr Gihan Gawish

12. Gel Electrophoresis
larger nucleic acids (greater than a few hundred bases)
the preferred matrix *agarose.
*Acrylamide, in contrast to *Polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning.
Dr Gihan Gawish

13. Gel Electrophoresis- Visualization
stained
Ethedium bromide
Silver
coomassie blue UV
Radioactivity
Photograph
Autoradiogram
Dr Gihan Gawish

14. Gel Electrophoresis- Visualization
If several mixtures have initially been injected next to each other, they will run parallel in individual lanes.
Incomplete separation of the components
overlapping bands
indistinguishable smears
multiple unresolved components.
Dr Gihan Gawish

15. Gel Electrophoresis- Visualization
The bands observed (unknown molecular weight) can be compared to those of the known (Molecular weight size markers) in order to determine their size.
Molecular weight size markers contain a mixture of molecules of known sizes.
marker run on one lane in the gel parallel to the unknown samples
Bands in different lanes that end up at the same distance from the top
contain molecules that passed through the gel with the same speed
means they are approximately the same size
Dr Gihan Gawish

16. Applications of Gel Electrophoresis
Forensics,
Molecular biology,
Genetics
Microbiology
Biochemistry.
The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device.
The image is recorded with a computer operated camera
Animation
Dr Gihan Gawish