ELECTROPHORESIS

Daheeya AlEnazi CLS 332

PRINCIPLES OF ELECTROPHORESIS

 Electrophoresis is a method used to separate charged particles

from one another based on differences in their migration speed
according in to there charge and size(m.wt).
 This is achieved by moving negatively charged molecules
(protein and nucleic acid) through a matrix (support medium )
with an electric field.
 Smaller molecules move faster and migrate farther than bigger
ones.
SUPPORT MEDIUM

1. a strip of paper or cellulose acetate (i.e. when small peptides

are to be separated).
2. Gel, such as Agarose and polyacrylamide (commonly used)
 ELECTROPHORESIS PARTS

 There are two electrodes (made of an inert metal, e.g. platinum) are immersed in two
separate buffer chambers.
 By using an electric power supply, electric potential (E) is generated between the two
electrodes.
 Charged particles migrate from one chamber to the other according to it charge.
 Negatively charged ions, called anions, move towards the positively charged anode,
while positively charged ions, called cations, move towards the negative charged
cathode.
 Different ions migrate at different speeds by their sizes and by the number of charges
they carry. As a result, different ions can be separated from each other by
electrophoresis
FORCES (F)

 A charged
  particle (+Q) moving in an electric field (E) in a non
conducting medium, such as water. Two forces are exerted on
the particle, one Fe, the force exerted on the charged particle by
the field, which is in the direction of the motion (toward the
cathode), and the other, Ff, the frictional force on the charged
particle, which retards its motion toward the cathode, and
hence is in the direction opposite to the motion (toward the
anode (+) electrode). This is shown in the diagram below:
CRITERIA OF GEL:

1. Hydrophilic
2. Chemically stable (not participate in chemical reactions during
electrophoresis)
3. Neutral (free of electric charges, otherwise it would act as an ion exchanger)
4. Mechanically resistant (should not be too elastic or too rigid as such gels
would be difficult to handle).
5. Bands need to be visualized in the gel by using special stain, the gel should be
transparent,
6. Pore size of the gel can be controlled during the preparation of the gel.
EFFECT OF PH ON MOBILITY

1. The effective charge of a substance varies with the pH also its

effective mobility varies.
2. Electrophoretic solutions must be buffered to carry the electric
field.
3. The buffer pH is chosen to give an optimum net charge for
maximum separation.
*AGAROSE GEL ELECTROPHORESIS

Mainly used in analysis or separation of DNA ,RNA molecules and

protein. Molecules move according their size & net charge.
Criteria:
Advantages:
Gel is easily poured, does not denature the samples and samples can
also be recovered.
Disadvantages :
gels can melt during electrophoresis, the buffer can become
exhausted, and different forms of genetic material may run in
unpredictable forms.
 PREPARATION OF GEL

A-Percentage of agarose %:
-Between 0.7% - 2%.
0.7%good separation or resolution of large 5–
10kb DNA fragments.
2%good resolution for small 0.2–1kb fragments.
Agarose dissolved in electrophoresis buffer.
B-Buffer:
Tris/Borate/EDTA (TBE)DNA
Tris/Acetic acid/EDTA protein
 ANALYSIS

After electrophoresis the gel is illuminated with an

ultraviolet lamp to view the DNA bands.
The ethidium bromide fluoresces reddish-orange in
the presence of DNA. (Carcinogenic stain )
photograph it with a digital camera.
Our lab:
Use program analysis to analyze the gel and take
picture of gel.
LAB PREPARATION

1. Make a 2% agarose solution in 100 ml TAE2g of agarose+100 ml of TAE

buffer.
2. put in microwave for 1- 1 ½ min . to dissolve the agarose.
3. Let it cool down to about55- 60 C at RT.
4. Add ethidium bromide stock in the gel solution for a final concentration of
0.5 ug/ml. Be very careful when handling the concentrated stock.
5. Stir the solution to disperse the ethidium bromide, then pour it into the gel
rack.
6. Insert the comb at one side of the gel, about 5-10 mm from the end of the gel.
7. Put gel into a tank with TAE. Gel must be completely covered with TAE, with
the slots at the end electrode that will have the negative current.
CONT.

 After the gel has been prepared, use a micropipette to inject about
3µl of stained DNA (a DNA ladder is also highly recommended).
 Close the lid of the electrophoresis chamber and apply current
(typically 100 V for 30 minutes).
 The colored dye in the DNA ladder (molecular weight markers) and
DNA samples acts as a "front wave" that runs faster than the DNA
itself. When the "front wave" approaches the end of the gel, the
current is stopped.
 The DNA is stained with EtBr, and is then visible under ultraviolet
light.
APPLICATION OF AGAROSE GEL

1. Separation Of DNA
2. Analysis of PCR products:
e.g. in molecular genetic diagnosis or genetic fingerprinting
PAGE
Daheeya AlEnazi
 POLYACRYLAMIDE GEL ELECTROPHORESIS

Technique widely used in biochemistry, genetics & molecular biology  to

separate biological macromolecules (proteins or nucleic acids) according
to their electrophoretic mobility.

Electrophoretic mobility is a function of the length, conformation and

charge of the molecule. 

Polyacrylamide gel with small pores helps to examine smaller molecules

better since the small molecules can enter the pores and travel through
the gel while large molecules get trapped at the pore openings.
ACRYLAMIDE

Acrylamide monomer is in a powder state before addition of water.

Acrylamide is toxic to the human nervous system, therefore all safety

measures must be followed when working with it.

Acrylamide is soluble in water and upon addition of water it

polymerizes resulting in formation of polyacrylamide.
 A-NATIVE-PAGE

Gel electrophoresis, molecules may be run in

their native state, preserving the molecules'
higher-order structure.

Protein structure
 B-SDS PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) :

is a method of separating molecules based on the difference of their molecular weight.
SDS chemical denaturant or detergent can destroys the protein secondary, tertiary or
quaternary structure and turning them into negatively charged linear polypeptide chains.

At certain pH  the SDS molecules are negatively charged and bind to proteins in a set
ratio, approximately one molecule of SDS for every 2 amino acids.
SDS is applies a negative charge to each protein in proportion to its mass. After running
the electric field ,the negatively charged polypeptide chains travel toward the anode with
different mobility.
The result:
The mobility of the molecules, is inversely proportional to the logarithm of
their molecular weight.

Use Tracking Dye (SDS dye):

To determined the distance traveled by molecule.
 TYPE OF DENATURANT

1. Urea For nucleic acids.

2. Sodium dodecyl sulfate (SDS)  for protein.

2-Mercaptoethanol(2MpE):
Reducing agent used to disrupt the disulfide bonds found between the protein
complexes, which helps further denature the protein.
 
GOOD PAGE

Using both Native and SDS-PAGE together can be used to purify and to
separate the various subunits of the protein. WHY??

Native-PAGE keeps the oligomeric form(quaternary protein structure) 

intact and will show a band on the gel that is representative of the level of
activity.
SDS-PAGE will denature and separate the oligomeric form into its
monomers, showing bands that are representative of their molecular
weights.
These bands can be used to identify and assess the purity of the protein
Procedure
 A- SAMPLE PREP.
*Prepare loading buffer by Adding:
1. SDS to break down the protein structure
2. Glycerol to give viscous medium to sample
3. Bromophenol blue Tracking dye (Sample dye gives blue color )
4. 2-MpE to break down the disulfide bonds of protein.

*Add 15 ul of loading buffer + 15 ul of protein sample.

*Mix in vortex, put in hot plate for 5 min at 95 C then centrifuge for 1
min
*then, samples are ready to load in to gel.
SAMPLE PREP.

2-mercaptoethanol
 B-PREPARING ACRYLAMIDE GELS

Gels consist of :

1. acrylamide, bis-acrylamide, denaturant (SDS or urea), and buffer
with an adjusted PH.(6.8-8.8)
2. Then, solution may be degassed under a vacuum or add butanol
(in case of protein)to prevent the formation of air bubbles during
polymerization and get smooth surface.
3. Ammonium persulfate and TEMED as source of free radicals
and a stabilizer and to initiate polymerization.
POLYMERIZATION REACTION

React 1 acrylamide with 35 bis-acrylamide, which can form cross-links

between two acrylamide molecules.
Acrylamide concentration from 5% - 25%.
Lower % better for resolving very high M.wt., while higher % of acrylamide
are needed to resolve smaller proteins.
POLYMERIZATION STEPS

8.8 for separation gel

6.8 for stacking gel
 Add separation and stacking gel

First layer:
Separation gel (7-15% acrylamide),PH=8.8 good for separation the molecules.
Second layer:
Isopropanol or ethanol to make flat surface. Put for sec. then, pour out.
Third layer:
Stacking gel(2-4% acrylamide),PH=6.8 use this PH because the N-terminal amino group
of the proteins and amino acids are protonated at equilibrium which makes them less
negative. The average electrophoretic mobility is very slow.
The result is line up all samples in same level.
After polymerizing put the gel between two glass plates in a gel caster, with a comb
inserted at the top to create the sample wells. After the gel is polymerized the comb can
be removed and the gel is ready for electrophoresis
DISTRIBUTION OF GELS
 START ELECTROPHORESIS

1. Put SDS dye. In 1st well

2. Load the samples.
3. Put the running buffer.
4. Start the electrical field for 5 min. & 50 Volt to line up all the samples at same
level.(Function of Stacking gel)
5. then, adjust the machine into 40 min & 150 Volt to start separation.
6. Fixation the gel( methanol + acetic acid) for 15 min.
7. Stain with Colloidal Coomassie stain for 1 hr.
8. De-stain with 100 DW for 15 min then change the water. Repeat for 4 times or keep
for overnight.
9. Take pic. by software.
The gel:
Either ventricle for protein or horizontal for DNA and RNA.
 FINAL RESULT

Bromophenol blue SDS Dye