appendix 12c

ElectrophoresisA.O. OBILANA CPUT

Electrophoresis Definition:

•Electrophoresis is a separation technique based on the differential transportation of charged species in an electric field through a conductive medium.

•Primary candidates for CE separation are ions.

Electrophoresis Definition:

•The separation of charged molecules using their different rates of migration in an electrical field.

Electrophoresis Definition:A technique to separate the DNA, ribonucleic acid (RNA), or protein molecules by using an electric current applied to a gel matrix

DNA is negative‐charged due to the sugar-phosphate backbone.

So it migrates from negative to positive electrodes through the gel matrix.

Electrophoresis Explained:

•Forced migration of charged particles (proteins, nucleic acids) in an electric field

•Fel = qE, where q is the charge, E is the applied electric field

•Molecule accelerates rapidly and is soon retarded by a frictional force

•Ffrict = -fv, where f is the friction coefficient, and v is the velocity

Electrophoresis Explained:

•After reaching equilibrium, qE + fv = 0 or ▫V = qE/f

•We define electrophoretic mobility as▫U = v/E = q/f

•U is an intrinsic property of the macromolecule, depending only on its charge and frictional properties, which is quite useful for characterization

Electrophoresis Equipment:

•Electrical Transformer : AC to DC

•Electrophoresis Tank : Flat BedColumnSlabCapillary

Electrophoresis Equipment:Flat Bed Electrophoresis Tank

Electrophoresis Equipment:Column and Vertical Slab Gel Electrophoresis tanks

ElectrophoresisFactors Influencing Separation – 1• Charge Density on Molecules – Difference

between media pH and pI of moleculeRole of Buffer• pH - Will influence charge density of a

protein and consequently the rate and direction of its movement.

• Ionic Strength - Influence rate of separation. • Composition - May interact with a protein

causing a change in the charge density. i.e. Borate ions and glycoproteins.

ElectrophoresisFactors Influencing Separation – 2

•Molecular Size and Shape•Nature of Support Medium

▫Restrictions on mobility▫Effect on Diffusion▫Electroendosmosis

•Micro-heterogeneity of molecular species

ElectrophoresisZonal Support Media

•Paper (historical)•Cellulose Acetate•Agar or Agarose•Starch (historical)•Polyacrylamide

Electrophoresis

Detection Methods• Protein stains

Amido Black Coomassie blue Silver

• Carbohydrate Stain Schiff's Reagent

• Lipid Stain Sudan black

• Histochemical Stains• Immunochemical Detection

Agarose gel electrophoresis:

•A method used in biochemistry and molecular biology to separate DNA or RNA molecules by size.

•This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).

•Shorter molecules move faster and migrate farther than longer ones.


Factors affecting Migration

1. DNA or RNA Molecular Weight2. Voltage3. Agarose4. Buffer5. Visualisation



1. DNA or RNA Molecular Weight•The length of the DNA molecule is the

most important factor, smaller molecules travel farther.



2. Voltage• The higher the voltage, the faster the DNA

moves. But voltage is limited by the fact that it heats and ultimately causes the gel to melt.

• High voltages also decrease the resolution (above about 5 to 8 V/cm)

Agarose gel electrophoresis:Factors affecting Migration

3. Agarose• Agarose gel electrophoresis can be used for the

separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus.

• Increasing the agarose concentration of a gel reduces the migration speed and enables separation of smaller DNA molecules.

• The distance between DNA bands of a given length is determined by the percent agarose in the gel.



3. Agarose• In general lower concentrations of agarose are

better for larger molecules because they result in greater separation between bands that are close in size.

• The disadvantage of higher concentrations is the long run times (sometimes days).

• Instead high percentage, agarose gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.

Agarose gel electrophoresis:Factors affecting Migration

3. Agarose• Most agarose gels:1. 1% gels are common for many applications.2. 0.7%: good separation or resolution of large 5–

10kb DNA fragments3. 2% good resolution for small 0.2–1kb

fragments.4. Up to 3% can be used for separating very tiny

fragments but a vertical polyacrylamide gel is more appropriate in this case.



4. BufferThe most common buffers for agarose gel:•TAE: tris acetate EDTA•TBE: Tris/Borate/EDTA•SB: Sodium borate.



4. Buffer

•TAE has the lowest buffering capacity but provides the best resolution for larger DNA.

•This means a lower voltage and more time, but a better product.



5. Visualisation• The most common dye used to make DNA or RNA

bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr.

• It fluoresces under UV light when intercalated into DNA (or RNA).

• By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20ng DNA becomes distinctly visible.

• EtBr is a known carcinogen, however, and safer alternatives are available.

Agarose gel electrophoresis:Equipment

Agarose gel electrophoresis:Advantages and Disadvantages

• The advantages are that the gel is easily poured, does not denature the samples.

• The samples can also be recovered.

• The disadvantages are that gels can melt during electrophoresis, the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.

• After the experiment is finished, the resulting gel can be stored in a plastic bag in refrigerator.

Electrophoresis (SDS PAGE)

•Sodium DodecylSulfate PolyAcrylamide Gel Electrophoresis

•Denature the protein in detergent•SDS is highly charged (-) and binds to

proteins in proportion to their molecular weight.

Electrophoresis (SDS PAGE)Structure of Polyacrylamide Gel


Systems used in Acrylamide Gel Electrophoresis

• Flat Bed• Rod or Column• Vertical Slab• Capillary


Factors Influencing Resolution -1• Polyacrylamide Gel Concentration

▫ Concentration of Monomer - is inversely proportional to the porosity of the final gel.

▫ Percentage of Cross-linker to Monomer – Has influence on the porosity of the final gel

▫ Chemical nature of Cross-linker – Can influence porosity and subsequent handling properties of the final gel.

▫ Purity of Monomer - Often contaminated with acrylic acid which causes electroendosmosis effects in the final gel.

Electrophoresis (SDS PAGE)Factors Influencing Resolution -2Buffer•pH.•Use of continuous or discontinuous

buffer systems.•Buffer composition•Inclusion of dissociation agents –

▫Urea,▫Non ionic detergents▫Sodium Dodecyl Sulphate (SDS)


Buffer SystemsDiscontinuous•These buffer systems can be used with or

without SDS although SDS inclusion is most common these days.

•Laemmli Buffer - Tris, Glycine HCl buffer – Most widespread used buffer especially with SDS.


Buffer SystemsDiscontinuous•Resolution is poor with MW sizes below

10KDa•Schaegger & Von Jagow Buffer system -

Tricine, Tris, HCl buffer - With right gel composition this buffer system can resolve peptides down to 1KDa and generally gives a better resolution up to 100KDa


Use of Dissociation Agents

•Sodium Dodecyl Sulphate (SDS)▫High Resolution Separation▫Molecular Weight Estimation

•Urea or Non Ionic Detergents (Tween 20)▫Help solubilize material otherwise

insoluble in aqueous media i.e. Cell membrane proteins

Electrophoresis (SDS PAGE)Detection of Components after Electrophoretic

Separation• Proteins

▫Amido black > 500 ng▫Coomassie blue 200-500ng▫Silver stain 20-50ng

• Glycoproteins▫Schiffs Reagent (Neutral Glycoprotein)▫Alcian blue (Acidic mucopolysaccharides)

• Lipid▫Sudan black

Electrophoresis (SDS PAGE)Detection of Components after

Electrophoretic Separation

•Enzymes▫Appropriate Histochemical methods

•Radiolabelled Proteins▫Autoradiography▫Flurography

•Immunochemical▫After Blotting onto Nitrocellulose paper


•SDS molecules exert strong internal repulsive forces that tend to stretch out the random coil protein into a rod-like shape

▫In essence, all proteins are made to look virtually the same, rods of the same diameter but with lengths that are proportional to the molecular weight of the protein.

Electrophoresis (SDS PAGE) – In Practice

• Denatured SDS-protein mixture with a colored dye or stain added for tracking is loaded at the top of a slab or tube of a gel (typically polyacrylamide)

• Electric field imposed within the gel using electrodes attached to a power supply▫ Proteins and dye migrate down the gel at a

constant rate that depends on the molecular weight of the protein

▫ Smaller proteins migrating faster


• At a given concentration of gel material and given electric field strength, standards of known

• molecular weight are used to empirically construct a calibration curve of molecular weight vs. electrophoretic mobility▫ Over a limited molecular weight range, the

electrophoretic mobility of proteins is found to be proportional to the logarithm of their molecular weight

▫ Rapidly and cheaply measure molecular weights with an accuracy of about 5% and can also determine trace amounts of impurities in a sample


• Samples are electrophoresed (native or SDS PAGE) through a cylindrical gel

• Bands pass through a thin frit within the elution chamber

• Isolated bands are drawn radially by a pump onto a fraction collector into discreet liquid fractions

Capillary electrophoresis (CE)

•Small-diameter capillaries (50 μm inner diameter, 0.5-1m length) allow use of very high electric fields

•Efficiently dissipate heat•Increasing electric fields

▫produces efficient separations▫reduces separation times

•20-30kV applied potential


• Electroosmotic flow▫ Surface of the silicate glass capillary contains

negatively-charged functional groups that attract positively-charged counterions

▫ Positively-charged ions migrate towards the negative electrode and carry solvent molecules in the same direction

▫ Uncharged molecules move at the same velocity as the electroosmotic flow (with very little separation). Positively-charged ions move faster and negatively-charged ions move slower, but all migrate towards cathode


•Small volume sample (10nL) injected at anode end, and detected near cathode end▫Detection similar to HPLC, includes

absorbance, fluorescence, electrochemistry, MS

•Cathode can be filled with gel, which eliminates electroosmotic flow▫Higher resolution, greater sensitivity, on-

line detection


Applications

•Analysis of optical impurities (chiral analysis)

•Tryptic digest analysis of recombinant biopharmaceutical drugs (peptide mapping)

•DNA analysis (e.g., PCR product analysis)•Organic acid analysis (e.g., in beverages)

ANY QUESTIONS?

appendix 12c

Technology

electrophoresis equipment

electrophoresis definition

fieldinversion electrophoresis

electric field electrophoresis

agarose gels

agarose concentration

percent agarose

dc electrophoresis tank