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Electrophoresis Methods
Electrophoresis Methods
Electrophoresis Methods
Electrophoresis Methods
BIOCHEM ISTRY FOR M EDICS 19/12/2021
Omeed A. ALI
Biochemistry Lab
Electrophoresis
• Definition
Electrophoresis is the migration of charged particles such as proteins dissolved in a
buffer on, or in, a supporting medium when an electric current is passed through them.
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Instruments and
Reagents
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Power supply
Tank ( Buffer ,Supporting media or Gel)
Detection and Quantification
Gel electrophoresis
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is a technique used to separate charged molecule (DNA, RNA and protein) under the
influence of an electrical field.
Molecules move at different rates according to their weight (mass) and charge:
1- Negatively charged molecules migrates toward
cathode (+) electrode
Positively charged molecule migrates toward anode (-) electrode
2- Small size (low M.wt) molecules migrates faster.
Large size (high M.wt) molecules migrates slower.
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Electrophoresis can be:
Horizontal or slab gel
Gel is poured in plat
Vertical
Gel poured between two
glasses
EQUIPMENTS AND MATERIALS
EQUIPMENTS AND MATERIALS
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Material (chemicals)
Supporting material (gel)
Medium (buffer)
Dyes
Sample
Marker
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1- Supporting medium:
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Paper (filter paper)
Cellulose acetate
Starch gel
Agarose gel
Polyacrylamide gel electrophoresis
(PAGE)
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Agarose and PAGE are commonly used.
Agarose gel:
porous material that sample move though
it.
Used in DNA and protein
Low range of conc. can prepared (0.5-3%)
Polyacrylamide gel electrophoresis (PAGE):
Is also porous consists of two material:
acrylamide + bisacrylamide
(cross link)
Used in: DNA sequencing, protein,
assessing M.wt of protein.
High range of conc. can prepared: (2-20%)
giving small pore size.
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Function: carry electric current.
Ionic strength = concentration of ions in solution.
When ionic strength of the buffer increased this lead to form sharp zones, but decrease
the migration rate.
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2- Buffer (ionic strength, pH)
3- Dyes & Visualization
Two types of dye are used:
1- Tracking dye or loading dye:
Used to monitor the migration, help in sample
loading.
Bromophenol blue.
2- Visualization dye:
Ethidium bromide (DNA, RNA).
Silver.
Coomassie blue dye (protein).
Ethidium
bromide
Silver staining
coonassie blue
dye
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4- Molecular weight size marker
• mixture of molecules of known sizes may be protein (Da) or DNA (bp).
Equipments
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Casting tray
Tank with cover
Comb
Power supply
Determination of Molecular Weight
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using PAGE of proteins or agarose gel for DNA
known M.wt (marker) is used along with sample
Run electrophoresis.
plot a standard curve of distance migrated vs. log Mwt
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procedure
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Sample
preparation
Gel loading
Gel running
Protein staining
Quantification
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