ANS 506: Electrophoresis
I. Introduction
1. Definition
A. The migration of a charged colloidal particle under
the influence of an electric field
a. Usually larger particles and/or molecules
B. Ionophoresis is used to designate the movement
of small ions
in an electric field
C. Form of chromatography
2. Used to Separate Molecules
A. Charge
a. Usually negative charge
b. Migrate (-) to (+)
B. Size
a. Molecular Weight
C. Shape
a. Secondary and Tertiary Structure
II. Classifications
1. Moving Boundary Electrophoresis
A. Features
a. No solid support
b. Molecules are dissolved or suspended in liquid buffer
c. Migrate in the liquid
B. Old Method
a. First described in 1886 (Lodge)
i. Used Dyes
ii. Dye migration still used as markers
in more modern electrophoretic
techniques
b. Used primarily in separating components
biological fluids
c. Also used in water purification
d. Refined by Tiselius (1925)
i. Very instrumental in study dissolved
proteins
ii. Optical system follows movement
of boundaries
--Measures refractive index
of a colorless solution
C. Advantages
a. Excellent degree of purity
b. Application to a wide variety
of high molecular weight substances
D. Disadvantages
a. Cannot separate individual components
b. Doesn't separate size and shape as well
c. Need large quantities
2. Zone Electrophoresis
A. Features
a. A solid support is used
i. Inert and homogeneous solid
ii. Starch Block, Agar Block, Cellulose acetate strips, Paper strips
b. Thin zone of material is fractionated
into components of different electrical
mobilities
B. Advantages
a. Individual components can be separated
b. Useful with small peptides and amino acids
C. Disadvantages
a. Harder purify components from solid than gel
b. Doesn't separate size and shape as well
as gel
3. Gel Electrophoresis
A. Originally grouped under Zone Electrophoresis
B. Features
a. Gel has a pores that can separate molecules
by size and shape
b. Most popular method for separation
of proteins and nucleic acids
c. Migration (distance) is inversely
proportional to the log of the size (MW, bp)
i. Always include molecular weight
or bp markers
d. Many applications in biochemistry, cell
and molecular biology, medical diagnostics,
forensics, etc.
i. Southern, Northern,
and Western Blotting
ii. DNA Sequencing
iii. DNA Fingerprinting
iv. Peptide Mapping
v. Isoelectric focusing
vi. More......................
C. Types
a. Starch Gel
i. Starch grains partially hydrolyzed
so that a "pore-like" gel
is obtained
ii. Little means of controlling
or changing pore size
b. Polyacrylamide gel (PAGE)
i. An artificial gel obtained
by a free-radical polymerization
of two monomer subunits.
ii. The amount of branching and pore size
can be controlled.
iii. Used to separate nucleic acids
and proteins
c. Agarose gel
i. A complex sugar obtained
from sea weed
ii. Pore size can be controlled
by changing concentration of agarose
iii. Used primarily with separation
nucleic acids
D. Advantages
a. Excellent separation on basis of size, shape,
and charge
b. Pore size and the amount of crosslinking
can be controlled
c. Excellent resolution
d. Small quantities can be resolved
e. Separation is rapid (30 min to a few hours)
f. Apparatus is relatively simple to operate
g. Applicable to a wide range of biological
compounds of different charges, sizes,
and shapes
E. Disadvantages
a. Quantification difficult by itself
i. Increased when combined
with procedures using radiolabeled
or photometric markers
b. Acrylamide is a neurotoxin
III. Factors affecting electrophoretic migration
1. Characteristics of the ion or molecule itself
A. Charge
a. Relevant pK values dependent on pH
of electrolyte (buffer) solution
B. Size
C. Shape
D. Tendency to dissociate
E. Amphoteric Behavior
a. Acid or Base
2. Characteristics of the environment in which the molecule
or ion is being studied (buffer or support)
A. Electrolyte concentration
B. Ionic strength
C. pH
D. Hydrostatic equilibrium
E. Viscosity
F. Temperature
3. Characteristics of the applied field
A. Intensity of current
B. Purity of current
3. Characteristics of the applied electrical field
IV. Polyacrylamide Gel Electrophoresis
1. Used to separate proteins and nucleic acids
A. Protein Characterization and Purification
B. DNA Sequencing
C. RNase Protection Assays
D. DNA Shift Assays
2. Chemical Structure
A. Polymerization of monomers
B. Acrylamide (Forms long chains)
C. N,N'-methylene bisacrylamide (Forms Crosslinks)
3. Catalysts
A. Polymerization initiated by Ammonium Persulfate (AP)
a. Riboflavin also can be used
i. Reaction stimulated by light
B. N,N,N,N'-tetramethylethylenediamine (TEMED)
is usually added to accelerate the reaction
a. Other chemicals also can be used
C. Polymerization is inhibited by low pH and oxygen
D. Rate of polymerization increased by increased
concentrations of TEMED or AP
4. Effective Pore Size
A. Increases as % of acrylamide increases
B. Bis- is primary determinate of pore size
a. Usually sold as 30:1 or 15:1
acrylamide to bis- ratio
C. 7.5% is a good place to start
5. Experimental Systems
A. Rod vs. Slab Gel
a. Vertical Slab most popular
b. Rod has advantages in large volumes
and 2 dimensional gel electrophoresis
B. Dissociating vs. Nondissociating buffer systems
a. Dissociating breaks molecules down into
subunits
i. Sodium Dodecyl Sulfate (SDS) most
common dissociation agent
--Other ionic detergents can also
be used
ii. -mercaptoethanol (BME)
or Dithiothreitol (DTT)
also included
--Break sulfide bonds
iii. Urea also sometimes used
as dissociation agent
--Breaks hydrogen bonds
iv. Heating protein mixture in denaturant
before loadingon gel is necessary
b. Nondissociating keep secondary and tertiary
structure
C. Continuous vs. Discontinuous (Multiphasic)
buffer systems
a. Continuous
i. Constant buffer composition, pH,
and Pore size throughout gel
ii. Usually used with nucleic acids,
less common with proteins
iii. Works best with small volumes
b. Discontinuous
i. Common method with proteins
ii. Used to separate larger volumes
iii. Discontinuities in buffer
composition and pH
--Manipulates pKs of molecules to
get better separations
iv. Stacking gel with large pore size
(top phase)
--Proteins stack at interface with
resolving gel
v. Resolving gel with smaller pore size
(bottom phase)
6. Processing after electrophoresis
A. Leave molecules in gel
a. Stain
i. Proteins--Commassie Blue,
Silver Stain
ii. Nucleic Acids--Ethidium Bromide
b. Expose to X-Ray Film
i. When radioactive markers are used
--DNA sequencing
--RNase Protection
B. Remove band (molecule) of interest
a. Purification of molecule
b. Whole lanes can be removed for
two dimensional gel electrophoresis
C. Transfer to Solids Support Filter
a. Nitrocellulose or Nylon (Blotting)
i. Southern Blot--DNA
ii. Northern Blot--RNA
iii. Western Blot--Protein
b. Electrophoretic transfer most common
c. Probed with DNA, RNA or Protein markers;
or antibodies
V. Agarose Gel Electrophoresis
1. Common method for separation of nucleic acids
A. Preparative Gels
B. Northern Blots
C. Southern Blots
D. Apoptosis "ladder" assays
E. DNA mapping
2. Agarose is heated and dissolved in appropriate buffer
A. The higher the Agarose % the better the resolution
a. 1% agarose common
B. Common buffers for DNA
a. Tris-acetate
b. Tris-borate
c. Tris-phosphate
d. Alkaline (NaOH)
C. Common buffers for RNA
a. Formaldehyde/MOPS
i. Most common
b. Glyoxal/DMSO
3. Advantages
A. Generally easier and faster to set up than PAGE
B. Resolution adequate for many situations
4. Disadvantages
A. Resolution not as good
VI. Other Electrophoretic Methods
1. Isoelectric Focusing
A. Used to fractionate molecules only differing
in net charge
B. Electrophoresis within a pH gradient
a. Molecule stops where net charge is 0
--Isoelectric point
C. Used in further purification of molecules
3. Two-Dimensional Gel Electrophoresis
A. Separate by one property in one dimension
B. Separate by another property in the other
C. Examples
a. Size and isoelectric point
b. Intact molecule and denatured
4. Immunoelectrophoresis
A. Characterization of proteins or other antigens
by gel migration and immunologic properties
B. Gel contains antibody
C. Antigen bound by antibodies
a. Form precipitation lines
5. Paper Electrophoresis
A. Used to separate small molecules
a. Amino acids
b. Small peptides
c. Nucleotides
B. High voltage used
6. Others................
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~~~~~Revised 6/26/97~~~~~ TAW