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

Velocity(v) of movement of a charged molecule in electrical field E

F=fv=qEv= 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/6R R : particle radius : Solvent viscosity Seperation by Size & net charge

F=fv f : frictional coefficient due to flow of solvent around moving object v : velocity of objectFor spheric molecule with radius R f= 6R (Stoke’s law) R : particle radius : Solvent viscosity

Transport of molecule in solution

In Electrophoresis….

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

Distance from origin, cm

Log MW

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

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)

DNA/Agarose Gel Electrophoresis

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 gelSuspend 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 mAAgarose 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)

AcrylamideBis-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 casting- percentage

acrylamideTotal percentage of acrylamide- acrylamide and bis-acrylamide- 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 casting- modifications

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

+ - + - + -Gel

Tank Electrodes Running buffer

Samples Sample Wells

Direction of migration

Loading Sample

Dye front

Running the Gel Run Complete

+ -+ - + -+ - + -+ -Gel

Tank Electrodes Running buffer

Samples Sample Wells

Direction of migration

Loading Sample

Dye front

Running the Gel Run Complete

+ + +- - -

Problems with ‘native’ PAGE

1. Oligomeric proteins2. Charge Density3. Shape

‘Native’ PAGE

High M.W.

Low M.W.

SDS-PAGE : separation by size

Denaturing method relying on two components- SDS and reducing agents

Sample is treated with both SDS and DTT or -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 background- methanol/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)

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

pH gradient in gelby ampholyte

pH

pI

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.

ApplicationAnalysis 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

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

Principles of Gel FiltrationVoid

volume

Elution Volume

Total Volume

Distribution

Constant

Molecular Weight Determinatin by GFC

LogMW

Kd

1. Determination of Vo : by Blue Dextran2. Calibration with Standard MW marker3. Sample Running & Ve Determination 4. Calculation of Kd5. 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 BasisArchimedes’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/2r = s(Sedimentation Coefficient)

Svedberg Equation

f= RT/ND D : Diffusion Coefficient

s=m0(1- ρ)/(RT/ND) RTs=NDm0(1- ρ)

Mr=RTs/D(1- ρ) Svedberg equation

R : physical constantD,T, ρ : Experimental Conditions, : Intrinsic physical properties of particle

F=fv=m0(1- ρ)rω2

Equipment

Outline design of AUC

Schemetic diagram of Beckman Optima XL-A

Experimental Set-Up

Assembly of Cell

Sedimentation Velocity Analysis

Apparent sedimentation coefficient

v=drb/dt = rb2s

Integrating

ln [rb(t)/rb(t0)]=2s(t-t0)

The velocity of sedimentation v is

We can calculate s valueJ=-D(dr/dc) J : diffusive flux c: conecntration of solute

D/s=M(1- ρ )/RTDetermination

of M

The behavior of particles as they move through the solvent during sedimentation at high centrifugal speed

Sedimentation Equilibrium AnalysisA 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)2](lncr)/ca[1/(r2-a2)]

cr : concentration of solute at distance rca : concentration of solute at meniscusa : 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 95/98Sedterp 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