Transcript Electrophoresis

Electrophoresis
Separation, analytical or preparative, of charged molecules by migration
through a matrix due to application of an electric field, with net movement
towards electrode of opposite charge
General Principle
The rate of movement depends on the field strength and
the number of charges. Biomolecules such as proteins
possess surface charge due to the presence of acidic and
basic amino acids
Transport of molecule in solution
F=fv
f : frictional coefficient due to flow of solvent around moving object
v : velocity of object
For spheric molecule with radius R
f= 6pR (Stoke’s law)
R : particle radius
 : Solvent viscosity
In Electrophoresis….
Velocity(v) of movement of a charged molecule in electrical field E
F=fv=qE
v= Eq/f f : frictional coefficient
q : net charge of on the molecule
Electophoretic mobility of a sample
U=v/E=Eq/Ef=q/f= q/6pR
R : particle radius
 : Solvent viscosity
 Seperation by Size & net charge
Separation can be effected by either or
both major components
• Size/shape
• Charge
• Both size/shape and charge
• Electrophoresis is not always run to endpoint- if molecules
are detected in matrix, empirical endpoint chosen such that
all molecules still in matrix
Estimating molecular size by
determining electrophoretic mobility
Mobility, Mr = distance migrated by band of
interest/distance migrated by dye front
Mr is related to logMW or log of molecular size in bp
in a linear fashion, therefore plot standards and can
determine sizes of unknowns
Estimating molecular size by determining
electrophoretic mobility
Log
MW
Distance from origin, cm
Classification
•DNA/agarose- horizontal; separation by size/shape
•RNA/agarose- horizontal; denaturing; separation by size
•Protein/polyacrylamide- vertical
–separation by size- SDS-PAGE (denaturing)
–separation by size and charge-native (non-denaturing)
–separation by charge (pI)- isoelectric focusing
–separation by charge, then size- 2D PAGE
(denaturing)
–Capillary Electrophoresis
DNA/Agarose Gel Electrophoresis
Horizontal submarine electrophoresis most
common; simplest separation by size
Agarose concentration 0.3-3%
Buffer most often Tris-Borate-EDTA (TBE) at 1X
or 0.5X; sometimes Tris-Acetate-EDTA (TAE) at
1X (recipes- Maniatis, Current Protocols)
Detection of DNA is generally by ethidium
bromide intercalation (dye in gel, in buffer, in
sample, or in immersion solution after run) or by
other dyes (e.g., Sypro)
Agarose
Good separation of all but smallest DNAs- mid-range to
large pore size provides sieving effect; relatively inert
Linear polysaccharide derived from seaweed extract;
composed of repeating units of agarobiose
When dissolved by heat in aqueous solution, then cooled, agarose
solution gels due to formation of inter- and intra-chain H bonds
=> The higher the concentration, the smaller the pore size
Preparing and running an agarose gel
Suspend agarose in running buffer (NOT H2O) to desired concentration
Heat to boiling; once dissolved, cool to ~65oC; add EtBr if desired to 1 µg/ml;
pour into gel tray with comb to form wells; let set completely
Prepare DNA samples- add loading dye to 1X (provides high density to allow
sample to sink, and provides dye for monitoring migration)
Remove comb from gel, set up in tank and submerge in buffer
Load samples by pipetting slowly through buffer into wells
Attach leads to tank and power supply; set V so I < 60 mA
Agarose concentration needed is determined by sizes of DNA fragments to be
separated
o for fragments between 0.3 and 3 kb, 0.7-1% are good standard
concentrations
o to separate small fragments (0.2-0.7 kb) from one another, use 1.5-3%
(smaller pore size)
o for separation of larger fragments (>3 kb and less than 30 kb) use 0.3-0.5%
Polyacrylamide Gel Electrophoresis
(PAGE)
Acrylamide
Bis-acrylamide + TEMED
Polyacrylamide
Disc(Discontinuous) PAGE
the Cl- ions move ahead of the
proteins and glycine as the glycine is
only partially dissociated at pH 6.7 
high conductivity, a low voltage gradient
in front of the protein.
The slower moving glycine ions result
in a region of low conductivity, hence a
high voltage gradient behind the proteins.
Protein stacking between the
chloride and glycine ions
Polymerization of acrylamide-Reaction
Polymerization is catalyzed by addition of
catalysts- ammonium persulfate and TEMED
TEMED catalyzes sulfate radical (SO4-.)
formation from persulfate; sulfate radical
attacks acrylamide monomers, which then
polymerize
Polyacrylamide gel castingpercentage acrylamide
Total percentage of acrylamide- acrylamide and bisacrylamide- determines pore size of gel
Discontinuous (disc) gels are most common for highest
resolution:
Low percentage (3% is usual) and low pH (6.8) are
used for stacking gel- all proteins run readily through
until hit higher percentage and pH (8.6) of running or
separating gel (4-20%), then stack up due to change in
pH before migration into running gel and separation by
charge and/or size
Polyacrylamide gel castingmodifications
 Gradient gels offer highest resolution from complex mixtures and for two or
more proteins with similar mobility
Often 4-20%, although several other percentage ranges
are used comonly
Migration of proteins slows considerably as reach small
pore size relative to protein size -> sharp banding
 Precast gels are available in many formats, including gradient
• Reproducibility
• Cost-effective if not too many used/day
• Convenient
‘Native’ PAGE
+-
Samples
+ -
Sample
Wells
+ -+
Gel
Tank
Direction of
migration
++-
Electrodes Running
buffer
High M.W.
Low M.W.
Dye
front
Loading Sample
Running the Gel
Problems with ‘native’ PAGE
1. Oligomeric proteins
2. Charge Density
3. Shape
Run Complete
SDS-PAGE : separation by size
Denaturing method relying on two components- SDS and reducing agents
Sample is treated with both SDS and DTT or b-ME at elevated
temperature; reducing agent ensures all disulfide bonds are reduced and
SDS denatures and coats protein with basically uniform charge density
(~1.4g SDS/g protein)- native charge masked and native shape lost so
separation primarily by size
Again, linear relationship of logMW and Mr allows MW estimation from
comparison with standard curve
Detection of protein using
Coomassie Staining
Coomassie Brilliant Blue is a common stain for protein gels. Staining is
carried out in methanol + acetic acid, which acts to fix proteins in gel.
Staining is carried out hours to overnight, depending on protein amount
and gel thickness; destaining is required to reduce backgroundmethanol/acetic acid without dye.
Coomassie binds to most proteins with similar affinity, but not all.
Binding is based on mostly ionic interaction (basic amino acids with SO3- on Coomassie) plus some hydrophobic interaction with Coomassie
rings.
Lower limit for protein band detection by Coomassie staining is ~10-100
ng.
Isoelectric Focussing(IEF)
pI
pH gradient in gel
by ampholyte
pH
In stable pH gradient in gel or capillary, each protein molecule
Migrate toward position of their pI.
At this point, their net charge is zero and migration stops
Ampholyte/pH Gradient Formation
a) Chemical Structure of
Ampholyte
b) Left : in the absence of
electric Field
Right : in the presence of
electric field
Capillary Electrophoresis (CE)
Disadvantages of traditional electrophoresis
•Long analysis times
•Lack of resolution
•Difficulties in detection
•Difficult to automate
Capillary electrophoresis
•Automated high-resolution approach to electrophoresis.
•Separation is carried out in a microbore fused-silica capillary,
around 25-75 µm internal diameter.
•The separation takes place is free solution and convection
currents are controlled by the capillary.
 Application
Analysis of proteins, nucleic acids, peptides, carbohydrates,
anions, cations, vitamins, organic acids, amino acids, pesticides,
even whole cells and viruses.
Equipment
Two Dimensional Electrophoresis
IEF or IPG (pI) + SDS-PAGE (Size)
Gel Filtration Chromatography
Separation based on size
(molecular exclusion or gel permeation chromatography)
Stationary phase : porous beads with a well-defined range
of pore sizes
Mobile phase : buffer/solvent containing sample to be
seperated
Principles of Gel Filtration
Void
volume
Elution
Volume
Distribution
Constant
Small proteins have access to the mobile phase inside the beads as well
as the mobile phase between beads and elute last.
Large proteins have access only to the mobile phase between the beads
and, therefore, elute first.
Proteins of intermediate size will then elute between the
large("excluded") and small ("totally included") proteins
Total
Volume
Molecular Weight Determinatin by GFC
LogMW
Kd
1. Determination of Vo : by Blue Dextran
2. Calibration with Standard MW marker
3. Sample Running & Ve Determination
4. Calculation of Kd
5. Calculation of MW
Gel Filtration Resin
Application
Purification of Protein
Buffer Exchange/Desalting
Molecular Size Determination
Sedimentation Method
:Analytical Ultracentrifugation
With Analytical
Ultracentifugation
the following protein
characteristics
can be determined:
Native Molecular Mass
Stoichiometry
Assembly Models
Conformation & Shape
Diffusion & Sedimentation
Assiociation
Native Molecular Mass
the only technique which you can determine
accurately the native molecular weight of a protein.
The obtained molecular weight is usually within 5%
of the
calculated value based on the protein sequence.
range of molecular weights :approx. 2.5 kDa up to
1.5 MDa.
A typical experiment takes about 16 hours
Stoichiometry
The stoichiometry of a protein complex can be
calculated from the determined molecular mass.
Assembly Models
The assembly of a protein complex can be calculted from the
determined molecular mass. It is even possible to follow the assembly
when the different partners are added to the mixture one by one.
 Ligand binding can also be analyzed using sedimentation velocity
methods if the ligand and acceptor differ greatly in their sedimentation
coefficients.
 Alternatively, a thermodynamic analysis may be made using
sedimentation equilibrium methods.
Conformation & Shape
Information about the shape and the conformation of a protein as well as the
interaction between macromolecules can be obtained from the sedimentation and
diffusion coefficients obtained from a sedimentation velocity experiment.
Sedimentation coefficients are particular useful for monitoring changes in
conformation of a protein. The resulting model for the overall shape of the protein
or protein complex can be compared with images obtained by electron microscopy
to assess how applicable those images are to the behaviour of these particles in
solution.
Assiociation
Unlike other methods for the study of binding processes, the sedimentation
equilibrium method is particularly senstive for the study of relatively weak
associations with associations constants (Ka) in the order of 10-100 M-1. However,
also binding processes with Ka values significantly greater than 107 M-1 can be
studied
Centrifugation:Physical Basis
Archimedes’s Principle
m=m0-m0 ρ
m : buoyant mass
 : partial specfic volume
ρ : solvent density
Centrifugal Force
F=mrω2 ω : angular velocity of rotation
Combing two equations
F=fv=m0(1- ρ)rω2
Multiplying by Avogadro’s number
M(1- ρ)/Nf = v/w2r = s(Sedimentation Coefficient)
Svedberg Equation
f= RT/ND D : Diffusion Coefficient
F=fv=m0(1- ρ)rω2
s=m0(1- ρ)/(RT/ND)  RTs=NDm0(1- ρ)
Mr=RTs/D(1- ρ)
Svedberg equation
R : physical constant
D,T, ρ : Experimental Condition
s,  : Intrinsic physical properties of particle
Equipment
Outline design of AUC
Schemetic diagram of
Beckman Optima XL-A
Experimental Set-Up
Assembly of Cell
Sedimentation Velocity Analysis
The behavior of particles as they move through the solvent during
sedimentation at high centrifugal speed
Apparent sedimentation coefficient
The velocity of sedimentation v is
v=drb/dt = rbw2s
Integrating
ln [rb(t)/rb(t0)]=w2s(t-t0)
J=-D(dr/dc) J : diffusive flux
c: conecntration of solute
We can calculate s value
D/s=M(1- ρ )/RT
Determination
of M
Sedimentation Equilibrium Analysis
A small volume of an initially uniform solution is centrifuged at a somewhat lower angular velocity
than required for a sedimentation velocity experiment. As the solute begins to sediment towards the
bottom of the cell and the concentration at the bottom increases, the process of diffussion opposes the
process of sedimentation. After an appropiate period of time, the two opposing forces reach
equilibrium
The flow of solute due to sedimentation
(black arrows) increases with radial distance.
This process is balanced at equilibrium by the
reverse flow from diffusion (open arrows)
M=[2RT/(1- ρ)w2](lncr)/ca[1/(r2-a2)]
cr : concentration of solute at distance r
ca : concentration of solute at meniscus
a : distance of the meniscus from axis of rotation
lncr vs. r2 plot
M(1-ρ)/2RT slope
Data Analysis
To analyze the data of a velocity analysis, there are 3 programs available:
Ultrascan on unix
SVEDBERG for Windows 95/98
dc/dt for Windows 95/98
Sedterp for Windows 95/98 for determination of the partial specific volume of
a protein and the density of the solute
For the equilibrium analysis
Windows 95 program called Ultrascan which is installed on the
XLA-pc.
 Nonlin for Windows 95/98 can be used to determine the association
constants for associating systems