Electrophoresis

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Electrophoresis

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Migration of charged solutes or particles in an electrical field

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Electrophoresis is a great method to

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separating charged biomolecules

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Components of electrophoresis

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• Driving force
• Support Medium
• Buffer
• Sample
• Detecting system

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Charged particles migrate towards

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the opposite charged electrode

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Velocity of migration is controlled by

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• Net charge of the particle
• size and shape of the particle
• strength of the electrical field
• chemical and physical properties of the supporting medium
• Electrophoretic temperature

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Cations migrate towards

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the negatively charged cathode

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Anions migrate toward

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the positively charged anode

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Neutral molecules

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are not attracted to either electrode

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Electrophoresis works because

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a particle with an electrical charge moves in a viscous medium by the force of an electrical field

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How is the driving force is counterbalanced

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The frictional resistance that the molecule must overcome to migrate in the support medium

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The velocity of a charged particle in a electrical field depends on:

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• (E) electrical field
• (q) particle charge
• inversely on counteraction forces

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Counteraction drag generated by

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viscous drag

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Equation for movement of charged particles in an electrical field:

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-V=Eq/f

V: velocity of the molecule

E: electrical field volts/cm

q: net molecule charge

f= friction coefficient , related to molecular mass and shape

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Support matrix types that proteins are applied to:

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At a single pH

• Cellulose acetate
• agarose
• starch
• polyacrylamide

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Conventional electrophoresis is:

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• Performed on gel paper
• minimal automation
• long analysis times
• detection performed post separation
• low voltages (because of heat build up)

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Electric current across the support causes the protein to:

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migrate at a constant velocity, based on their surface charge, size, and shape

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Power supply either run on:

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Constant voltage

Constant power

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Ohm's law

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V=IR

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Voltage

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current (amps) X resistance

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Constant voltage depends on:

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q/f (charge/resistance)

migration rate is proportional to the molecular charge to mass ratio

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Electrophoretic factors

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• Molecule charge
• molecular size and shape
• ionic strength
• buffer pH
• voltage
• temperature
• time
• diffusion (when power is off)
• wicking (evaporation)
• matrix
• electro-osmosis

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Acidic pH will give a molecule a

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positive charge

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Alkaline pH will give a molecule a

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negative charge

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Peltier device

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In automated electrophoresis:

conducts heat away from the gel to prevent protein denaturation

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Why are high quality reagents needed

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impurities may effect electrophoresis

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Buffer is responsible for:

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carrying the current

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The buffer pH determines

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The charge of the solutes and thus their migration

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Buffer ionic strength

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determines the electroendosomsis

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What happens if the gel runs too long?

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the solutes can run off the gel (except in IEF)

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Tracking dye

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bromophenol blue- small anionic compound moves the fastest

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Band diffusion

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separated bands tend to diffuse and broaden after the power supply is turned off

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How to stop protein diffusion

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fixing the gel

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How to remove unbound stain

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destain to produce a clear background

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What specimen should be used

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Serum- plasma has fibrinogen which produces an extra band

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Support media

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cellulose acetate or agarose gel

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Current and volatge

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constant

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Types of stains

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• Colormiteric
• Fluorescent
• Enzymatic
• Radioactive

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Protein Dyes

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• Amido black
• Ponceau S
• Bromophenol blue
• Coomassie brilliant blue

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Silver stains

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10-1000 times more sensitive than dyes:

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Protein dye interactions depends on

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• Type of protein
• Degree of protein denaturation

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Destains typically used:

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• 7-10% acetic acid
• water: methanol: acetic acid (5:5:1)

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Quantitation

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Performed by densitometry

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What is a densitometer

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reads electrophoresis pattern

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Parts of densitometer

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• Light source
• Filter system
• photo-detector
• converts electrical signal into numbers

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The denser the band

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the more light they absorb

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Tracing of the pattern of a gel and

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calculates the percentage of each fraction

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In-gel kinase

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Method to identify kinase substrate:

enzymes can still be active after electophoresis; the product of its substrate is visualizable

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Ethididium bromide

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interclataes between bases increases its fluorescent intensity

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Visualizing nucleic acid stains

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under uv light as red-orange stains on the gel

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Autoradiography

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• In a darkroom the gel is covered with a heavy chromatography paper
• Covered with plastic wrap
• Placed on an x-ray film
• triple sandwich is placed in a folder incubated at -70 to avoid band diffusion

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Biotin

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can be attached to large biomolecules; properties of the enzyme are measured

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What pH is used for protein separation

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over 8.0 for optimal separation:

proteins will carry a net negative charge

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Endosmosis

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movement of solvent and solutes in relation to the fixed support

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Electroendosmosis

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1. Neg charge on gel
2. Positive charge cations in the buffer move toward the cathode
3. Uncharged or weakly negative charged molecules are carried toward the cathode by the electroendosmotic movement

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What support media eliminate electroendosmosis?

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Neutral support media:

agarose

acrylamide

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Heat problems

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• Heat= volts X current X time
• current meets electrical resistance which causes heat build up
• Compensate by cooling gel or lower voltage

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Wicking Problems

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Heat can cause solvent to evaporate from the gel:

• wicking of the buffer onto the gel from both compartments to replace the solvent that evaporate
• flow of buffer from both directions affects migration

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Types of electrophoresis

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• Paper
• Starch gel
• Agarose gel
• Cellulose acetate
• Polyacrylamide
• disc gel
• IEF, IEP, IFE, CE

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Disc gel electrophoresis layers

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• Upper layer
• Lower layer

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Disc gel:

upper layer

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stacking gel

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Disc gel:

lower layer

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resolving gel

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Disc gel:

The buffers for two layers

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they are different ionic strength

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Disc gel:

Pore size of the two layers

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• Stacking gel: decreased concentration of acrylamide (larger pore size)
• Resolving gel: increased percentage of aryclamide (smaller pore size)

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The two layer format causes the proteins to:

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form highly concentrated bands: allows for greater resolving power

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PAGE

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polyacrylamide gel electrophoresis

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How does PAGE separate biomolecules

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size sieving and charge; small molecules move faster

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Chemical polymerization in PAGE is controlled by:

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free radical initiator-catalyst system- TEMED/Ammonium persulfate

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Photochemical polymerization is initiated by

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riboflavin in the presence of UV radiation

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The PAGE gel is formed by:

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the free radical polymerization of acrylamide and the cross-linking agent N,N'-methylene-bisacrylamide

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Acrylamide Reagents:

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All are toxic:

• Acrylamide
• N,N,N,N'-tetramethylethylenediamine (TEMED)
• N,N'-methyllene-bis-acrylamide
• Ammonium persulfate

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Resolving power of PAGE

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Depends on the concentration of acrylamide and bisacrylamide

• Lower concentration gives gels larger pore size
• Hi conc gives gels smaller pore size

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pH of PAGE

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basic;

most biomolecules neg charged and will migrate to the anode

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SDS-PAGE

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Separates biomolecules based on size:

• Proteins interact with the detergent of about 1%
• Produces a linear peptide chain

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SDS

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Sodium dodecyl sulfate

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SDS masks:

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the charge of the protein by forming anionic complexes; give proteins a net negative charge per unit mass

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SDS disrupts what bonding

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hydrogen

blocks hydrophobic interactions and partially unfolds proteins (eliminating secondary and tertiary structure

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SDS-Page can be further processed

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Western blot

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Nucleic acid gel:

Sample pretreatment

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• Nucleic acids are digesting into fragments of varying size with common ends
• Samples are treated with a denaturant (urea or formamdie) to prevent denaturation
• Bases are modified

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Nucleic acid gel electrophoresis

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Nucleic acid fragments with common ends are electrophoretically separated by PAGE:

Optimized by altering % acrylamide

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Nucleic acid detection

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• Labeled with 32P or a fluorescent tag
• Autoradiography: radioactive
• Fluorimeter: fluorscent tag

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Base pair range of DNA and RNA characterized

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200-50,000 bp

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DNA electrophoresis matrix

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Agarose, seaweed extract; serves as a sieve

Density dependent on the concentration

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DNA charge

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Net negative charge (due to phosphate group)

Migrates towards the anode

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Rate of DNA migration is dependent upon:

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• Size (bp length)
• Shape
• Agarose concentration
• voltage
• buffer composition
• dye

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DNA mobility

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• Supercoiled compact DNA migrates faster through the gel than linear or circular shapes
• Shorter fragments move faster than longer fragment

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DNA size determination

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• With linear DNA; migration rate is inversely related to the log of the DNA fragment length in bp

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DNA visualization

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• Ethididium bromide- fluorescent dye

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DNA fingerprint

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the electrophoretic patter or restriction pattern is a DNA fingerprint of fragments separated by size

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Pulsed field gel Electrophoresis (PFGE)

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Separates large DNA (200-300kb)

• Electric field changes repeatedly in direction and strenght
• The electrical pulses cause the gel to become more fluid changing DNA conformation; the DNA "slithers" through the gel matrix

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2-D electrophoresis

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• Different electrophoretic technique in each dimension
• Proteins separated by different properties

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2-D electrophoresis First dimension

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Proteins are separated by charge in IEF

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2-D electrophoresis second dimension

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Proteins are separated by size in SDS-PAGE

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2-D electrophoresis is used for

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Proteome analysis;

Proteom complements expresses by a genom

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Steps of 2-D electrophoresis

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1. Sample preparation
2. First dimension isoelectric focusing
3. Second dimension gel electrophoresis
4. Staining
5. Imaging analysis

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Immunoelectrophoresis (IEP)

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Two step process:

• First step separates protein on agarose gel electrophoresis
• Second step: proteins interact with specific antibodies
• examine the proteins' antigenic properties

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IEP- antibody interaction

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Antibody is added to a trough cut in the gel;

diffuses through the gel to separated protein

If there is a reaction a visible insoluble precipitin is formed

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IEP uses

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Assesses protein:

• Purity
• Composition
• Antigenic properties

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Immunofixation electorphoresis (IFE) involves

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protein electrophoresis followed by an immunoprecipitation

Wash stain and destain to visualize the protein:antibody complex

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Ampholytes

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synthesized polyamine carboxylic acids, multi-branched chained and deprotonated carboxylic acid groups

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Properties of Carrier Ampholytes

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• Maintain pH due to have a good buffering capacity
• Conduct electric current due to zwitterionic
• Net zero mobility at their pI's
• Soluble in water
• Do not interfere with the sample or its detection

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Use of Carrier ampholytes

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• Mixing a series of carrier ampholytes
• Apply them to a gel matrix ( acrylamide), pH gradient is generated when a electrical current is applied to the gel

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pH range of IEF is determined by

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the ampholyte mixture

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Amino acids are amphoteric because

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the carry multiple charges:

acidic pH: positive charge

alkaline pH: negative charge

the net charge on a protein changes as the pH changes

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What is IFE

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electrophoresis in a pH gradient, separation base on pI

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Isoelectric focusing

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as a protein approach their pI they gradually become less charged

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IEF set up

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A solution of ampholytes is mixed with the gel prior to polymerization

When an electrical field is applied the ampholytes move their pI to create a constant pH gradient (creates a buffer zone)

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Ampholytes movement

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the move ahead faster than the proteins because they are smaller est the pH gradient ahead of the protein

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IEF protein migration

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As the protein migrates through the different pH zones, its net charge decreases

Once the protein has no charge it cannot move

Stops when it enters the pH zone that equals its isoelectric point

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IEF

pH<pI

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protein carries a net positive charge and migrates towards the cathode

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IEF

pH>pI

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proteins carry a net negative charge and migrate towards the anode

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IFE

pH=pI

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protein carries a net zero charge does not migrate

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Protein bands in IEF

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Bands are very sharp due to the small pH ranges

• Relatively independent of electrophoresis duration
• Balance between focusing and diffusion
• Resolution will be within 0.2 pH units

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IEF protein visualization

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ampholytes must be removed to be stained:

Soak in 5% TCA solution

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Added benefit of IEF

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concentrates proteins because they are focused in a specific pH zone

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IEF uses

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• Estimate protein purity
• Determine protein identity by comparing migration with that of a known protein standard

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Capillary Electrophoresis

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Conventional Electrophoresis:

on gel or paper, minimal automation, long anaylsis, detection performed post separation, low voltages

Performed in capillary tubes, hi voltage can be used due to heat diffusion of the tubes

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Advantages of CE

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• Automation
• High voltage
• rapid real time separation
• in-line detection
• high separation efficiency
• Small sample size

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CE uses

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• Narrow bore fused silicia capillaries filled with buffers
• Sample loaded by replacing the buffer at the anode end
• electro-osmotic flow; bulk of liquid to cathode

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Applications of CE

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• Pharmaceuticals
• DNA
• Proteins, peptides
• Clinical and forensic specimens
• Agrochemicals
• Natural products
• Fine chemicals
• biochemicals