Electrophoresis

Önder Bozdoğan
Kırıkkale University Faculty of Medicine
Pathology Department
 Questions
• What is electrophoresis?
• History of electrophoresis?
• Types of electrophoresis
• What are the main principles of electrophoresis?
• What are the equipments and reagents of
electrophoresis?
• How can we pour an agarose gel?
• What is the meaning of the bands after electrophoresis?
• Is there an automated type of electrophoresis?
 Electrophoresis-Definition
• Electro=Electric; phoresis= Migration;Carry
accross.
• A kind of separation technique based on the
differential migration features of charged
molecules in an electric field.
• An analytical method frequently used in
molecular biology, biochemistry and medicine.
 History of electrophoresis
• 1834-Michael Faraday –“Faraday's laws of electrolysis”
• 1882-Robert Koch- Agarose culture medium
• 1937- Arne Tiselius “A New Apparatus for Electrophoretic Analysis of
Colloidal Mixtures"- Tiselius apparatus.
• 1946-Agarose gel
• 1955-Oliver Smithies-starch gels
• 1957-Joachim Kohn-Cellulose acetate electrophoresis
• 1959-Acrilamide gels
• 1969-Weber K and Osborn M. SDS gel electrophoresis
• 1971-Danna and Nathans-The separation of DNA by gel electrophoresis
• 1977-Sequencing gels.
• 1983-Pulsed field Electrohoresis
• 1983-Capillar electrophoresis
 Electrophoresis
• Principle:
– In an electrical field charged molecules and
particles migrate to the opposite charge.
– Usually in aqueous solution(Buffer).
– Due to their varying charges and masses, different
molecules and particles in the mixture are
migrate at different speeds.
– As a result; separated into single fractions(bands).
 Buffer
Main Components
_
Cathode Anode
+
- GEL
Sample
-
-

Electric Field
 Migration Depends on
• Strength of electric fields.
• Temperature
• Features of the molecule
– Net charge of molecule
– Size of molecule
– Shape of molecule
• Features of the Gel
– Gel type
– Gel concentration
• Buffer Type/pH.
 Electrophoresis
– Separates
– Nucleic acids
– Proteins
– Peptides
– Amino acids
– Organic acids/bases
– Drugs
– Pesticides
– Inorganic anions/cations.
– Everything that can carry a charge.!
 Molecular Pathology
• Nucleic acids.
– Determining quality of DNA/RNA
– Analyses of PCR products
– Mutation detection
– Southern and Northern blotting
– Sequencing
• Proteins
– Western blotting
– Protein purification
 Electrophoresis Types
• Gel electrophoresis
– Agarose gel
– Polyacrylamide gel
– Others.
• Pulsed Field Gel Electrophoresis
• Capillary Electrophoresis
• Isoelectric focusing
• 2D electrophoresis
 Gel Electrophoresis
• Use of a gelatinous material.
• The gel acts as a support medium
• Used to separate proteins or nucleic acids.
 Gel Types
• Starch-Rarely used
• Polyacrylamide-Protein, small nucleic acid
Commonly
fragments used

• Agarose-Nucleic acids, large proteins

• Cellulose acetate-Proteins
 Agarose Gel Electrophoresis
• Easy, fast, well established
method for separating DNA
fragments.
• Agarose, a polysaccharide
derived from seaweed.
• β-1,3-D-galactose/3,6-
anhydro-α-1,4-galactose.
• Dissolves in boiling water,
and hardens, becomes gel
*Sigma MSDS
when cooling.
• Bigger pore size than
polyacrylamide
 Agarose Gel Concentration/DNA-Size
Concentration Size
0,3 5000-60000 base
0,6 1000-20000 base
0,7 800-10000 base
0.9 500-7000 base
1.2 400-6000 base
1.5 200-3000 base
2.0 100-200 base

Temizkan G, Arda N. Moleküler biyolojide kullanılan yöntemler. Nobel tıp.

 Polyacrylamide Gel Electrophoresis
(PAGE)
• Synthetic polymer
• Formed from acrylamide
subunits.
• Acrylamide with a cross linker,
methylene bis-acrylamide .
• Polymerization catalysts:
• Ammonium persulfate (APS) +Tetramethylethylenediamine (TEMED )
• Light

• 3.5–20% concentration.
• High resolution. http://www.biocompare.com/Application-Notes/42631-Acrylamide-Polymerization-A-

Practical-Approach /
• Acrylamide is a dangerous
neurotoxin
 Buffer
• Provides ions in solution for electrical conductivity.
• Prevents the pH changing.
• Common using buffers:
• Tris Borate EDTA (TBE)-Stable, expensive,PAGE, long
separation time.
• Tris Acetate EDTA (TAE)-Inexpensive,short separation time.
• Tris Phosphate EDTA (TPE)
• RNA
– Sodium phosphate Buffer
– MOPS Buffer (-3-(N-morpholino) propanesulfonic acid)
*Buffer formulation
http://www.elabprotocols.com/
 Equipment
• Power supply
• Cooling Apparatus
• Electrophoresis gel apparatus-Vertical or Horizontal
• White Light/UV Light Box/Digital Camera/Gel Documentation
System
• Reagents:
• Gel staining chemicals(eg.EtBr)
• Prepared gels or gel chemicals
• Buffers
• Loading dyes

• Other laboratuary equipments:

– pH meter
– Pipettors
– Lab. Scale
– Stir plates
 Electrophoresis Gel Apparatus
Horizontal (Flat bed)
• Gel thickness limited.
• Only one gel per aparatus
• Easily adapts different
techniques.
• Technician friendly
• More safe for electricity
accidents.
• Gnll. used for agarose gel
electrophoresis
 Electrophoresis Gel Apparatus
Vertical
•Different gels thicknes can be
used.
•More than one gel per apparatus
•Not easily adapted for different
techniques.
•No technician friendly.
•Gnll. used for polyacrylamide gel
electrophoresis
Gel Documentation System
Gel Documentation System
UV/White Lamp Box
Power Supply
 Technique

• Steps
– Sample preparation
– Gel,buffers, etc. preparation.
– Load markers
– Load samples
– Running of the gel
– Staining of the gel
– Photography, gel documentation
– Interpret/analysis of gel
 Do not forget!
• DNA molecule is an organic acid.
• Negatively charged.
• Migrate toward the positive electrode(Anode)
in an electromagnetic field.
• Small fragments go further than large
fragments of DNA.
• Do not forget “Running of the gel “
• Cut off electricity before taking gel from
apparatus.
 Markers

Selection of the suitable marker for the expected fragment size is very
important.!
 Ethidium Bromide
• Powerful mutagen but it works well.
• Cheap, sensitive, easy to use, fast.
• Binds to DNA .
• Fluorescens under UV lamp and visualizes of
DNA on the Gel.
• Can be added directly into the gel and/or
buffer
or

• Gel can be stained after run.

• Concentration 0.5-1ug/ml for staining gels.
 EtBr Alternatives
• Sybr stains.
• Silver stains
• Methylen Blue
• Commercial stains.

• More safe, less sensitive.

• Syb stains also mutagenic.??
 Voltage

• More voltage, more quick gel runs.

• But,
– Low resolution.
– Increase temperature
• As a result, low quality separation.
• <5-8 V/cm of gel length 75mA.(100mA for
minigels)
• By trial and error (Emprical approach)
 Technique

• Sample preparation
• Gel,buffers, etc. preparation.
• Load markers
• Load samples
• Running of the gel
• Staining of the gel
• Photography, gel documentation
• Interpret/analysis of gel
 Technique

• Sample preparation
– PCR products
– DNA, cut with restriction enzymes
– Others
 TBE Buffer X10

Power Supply Microwave oven

Agarose

Distilled Water

Gel apparatus
 Gel,buffers, etc. preparation.

– Agarose, desired concentration.

• 100 ml .......gr.
– Solved in buffer.
– Melt in the microvave.
• Clear solution when melt.
– Wait a few minutes and add EtBr.
– Insert the” comb”
– Prepare apparratus, pour gel.
– Wait until the gel hardens
– Pull out the comb carefully.
– Agarose, desired
concentration.
• 100 ml .......gr.
– Dissolve in buffer.
– Swirl the solution
periodically.
– Melt in the microvave.
• Clear solution when melt.
• Be careful. The boiling
agarose solution may be so
hot.
 Comb

– Wait a few minutes for

cooling and add EtBr. EtBr

– Insert the” comb”

– Prepare apparratus, pour
gel.
– Wait until the gel
hardens(15-20min. )
control the gel
– Pull out the comb
carefully.
• Do not forget ! EtBr is a
powerful mutagen wear
gloves.
 Load markers/Load samples

• Load markers to first well.

• Load the samples mixed
with a dense loading dye.
• Be careful ! Not to
contaminate other wells
• Be careful! Not to
perforate the well.
• Loading dye includes:
• Bromophenol Blue
• Glycerol
• Other:Xylene Cyanol FF
,EDTA
 Running of the gel
• Control the anode and cathode
• The power source is turned on.
• Air bubbles!
• The gel is run.
• The time depends upon the
amount of current and % gel.
• Control the gel several times
• Track bromophenol blue.
(migrate near 300bp/%1,5
agarose)
 Photography, gel documentation
• Take off the gel.
• Stain if you do not add EtBr
before gel casting.
• The gel is then visualized by
UV light.
• Analyse the gel.
• You can also
– Cut the band on the
gel(DNA) for further
techniques.(e.g. Plasmid
studies)
– Remove from the gel( e.g.
Southern Blotting)
 TXNIP Primer selection.

Unexpected
band

Products

Primer Dimers
Kolon cell lines GAPDH
 Protein Electrophoresis
• Simple to use and highly reproducible
technique.
• Provide information of the molecular weight,
charged, subunits, purity of protein mixture.
• SDS-Page most common used technique.
– Native PAGE:
– Separates folded proteins by charge, size, and shape.
– Denaturing gel electrophoresis
– Separates folded proteins by size.
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
– SDS PAGE:
– Denaturing gel electrophoresis
– Give information of the size of polypeptide chains.
– Separated by length of their polypeptide chains not by its
charge.
– SDS binds to and unfolds the protein established a negative
charge.
– Without SDS proteins migrate charge mass ratio.
 Polyacrylamide Gel Electrophoresis
(PAGE)
pH=6,8 -
Buffer
Stacking Gel •Stacking Gel
•Low concentration
•4-5% acrylamide
Separation Gel •For concentrating
proteins
•Large pore size
•Separating(Resolving) gel
•High concentration
•5-20 % acrylamide
pH=8,8 •Smaller pore size

+
Buffer
 Acrilamide concentration/Molecular
weight
Concentration Molecular weight
15 12-43 kD
10 16-68
7,5 36-94
5 57-212

Temizkan G, Arda N. Moleküler biyolojide kullanılan yöntemler. Nobel tıp.

 Protein Electrophoresis
• Proteins in the gel stained by:
• Coomassie Blue dye
• Silver staining
• Others(Flourescence, commercial dyes)
 Other Protein Electrophoresis
Techniques
• IEF(Isoelectric focusing)
– Separates proteins by their isoelectric points (pI)
by using pH gradient of the gel.
• 2D PAGE(Two dimensional gel electrophoresis)
– Separates proteins are by two properties (eg: pI
and size) in a mixture.
• Western blotting:
– Separating proteins first by size then staining with
specific antibody-antigen reactions.
– Technique gives molecular weight and identifies
specific protein.
 Pulsed Field Gel Electrophoresis
(PFGE)
• Used for separating very - -
large DNA molecules.
(1Mb<)
• Based on the periodically
changes of directions in
the electric field.
+
- +-
• Gnll. used for genotyping.
• Gold standard in
epidemiological studies of
(Subtypes)pathogenic
organism + +
 Capillary Electrophoresis
• Process large number of samples than
classical techniques.
• Main technique first described by Hjerten in
1967.
• The first commercial CE instrument in 1988.
 Principle
• Power supply.
• The anode and cathode
buffer reservoirs with
corresponding
electrodes.
• The separation
chamber(capillary tube).
• The injection system.
• The detector
*GEORGE P. PATRINOS , WILHELM ANSORGE ; Molecular Diagnosis
 Capillary Electrophoresis
• Applications
– Analyzing proteins in physiological matrices
(eg.Serum, urine)
– DNA analysis
– Drug screening.
– Analysis of pesticides, food content, pollutants.
 Specific Applications
• Neoplastic disorders
– Detection of tumor-related mutation.
– Microsatellite instability
– Analysis of monoclonality.
• Diagnosis of hereditary diseases and prenatal
testing
• Diagnosis of infectious diseases
• Identity testing
 REFERENCES
• Temizkan G, Arda N. Eds. Moleküler Biyolojide Kullanılan Yöntemler. Nobel Tıp Kitapevi.2004.
• Patrinos G,Ansorge W. Eds.Molecular Diagnostics.Elsevier. 2005.
• A Guide to Polyacrylamide Gel Electrophoresis and Detection.BioRAD application booklet.
• Westermeier R. Electrophoresis in Practice. Wiley. 2005.
• Borst P. Ethidium DNA agarose gel electrophoresis: How it started. IUBMB Life (International Union of
Biochemistry and Molecular Biology: Life) 2005; 57: 745-747.
• Petersen J R.et al . Capillary electrophoresis and its application in the clinical laboratory. Clinica Chimica Acta 2003;
330: 1-30.
• Roberts GA, Dryden DTF. DNA Electrophoresis: Historical and Theoretical Perspectives. 2013;1054:1-9.
• Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis
2009;30(S1):S188-S95.
• Perrett D. 200 years of Electrophoresis. Chromatography Today .December 2010.
• Basım E. Basım H. Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology. Turk J Biol
25 (2001) 405-418.
• Prischmann J. Basics and Theory of Electrophoresis. www.seedtechnology.net/.../ELBasics2010.pdf.
• Gödde R. et al. Electrophoresis of DNA in human genetic diagnostics – state-of-the-art, alternatives and future
prospects. Electrophoresis 2006, 27, 939–946.
• Petersen JR. Capillary electrophoresis and its application in the clinical laboratory. Clinica Chimica Acta 330 (2003)
1 –30.
• Patricia Barril and Silvia Nates (2012). Introduction to Agarose and Polyacrylamide Gel Electrophoresis Matrices
with Respect to Their Detection Sensitivities, Gel Electrophoresis - Principles and Basics, Dr. Sameh Magdeldin
(Ed.), ISBN: 978-953-51-0458-2.

This presentation was prepared as a course handout.