Electrophoresis

Wide application in biochemistry for separation/analysis of eg amino acids, nucleotides, proteins, nucleic acids, proteoglycans etc.

Electrophoretic Mobility

Rate of migration depends on:

                                     MOVING FORCE ON ION = E.q
where  E = electric field strength (units of volt.cm^-1),
        q = charge on ion

                                     RETARDING FORCE ON ION = f.v
where  f = frictional coefficient - depends on size & shape of moving ion,
        v = velocity

Migrating ion attains constant velocity at which moving and retarding forces balance

ie                                                     v = ^E.q/_f

         ELECTROPHORETIC MOBILITY, m   = rate of movement at unit field strength

Why Use Electrophoresis?

Versatile separation method because:

• electrical charge on polyelectrolyte ions can be modified by change of pH
  
• even if species have same charge, differences in molecular size and conformation affect mobility because these factors determine f
  
• mild, non-denaturing technique for proteins (minimal or no interaction with surfaces)
  
• high sensitivity - small sample size

Method of choice for

• analysis of mixtures, and
  
• as a criterion of purity

Limited to water-soluble ionic species.

Chromatography usually better for preparative separation.

Moving Boundary Electrophoresis (Tiselius)

• Buffer throughout (characteristic of all electrophoresis) - conducts electric current and maintains constant pH, hence constant state of ionization
  
• Gives BOUNDARIES but not ZONES (zones would be gravitationally unstable leading to convection)
  
• Superceded by electrophoresis on supporting media - greater sensitivity and better separation (zones).
  
• Free solution electrophoresis (but zonal, not moving boundary) "reborn" as CAPILLARY ELECTROPHORESIS

Electrophoresis on Supporting Media

Inert, hydrophilic solid or gel-like support.
Migrating molecules move through microscopic channels.

Advantages

• Less prone to convective disturbance (bulk flow of solvent impeded)
  -----> ZONAL electrophoresis achieved
  
• Molecular sieving and/or adsorption effects increase separation possibilities
  
• Can choose different support material for different applications
  
• Sensitive. Detect µgram amounts in separated zones using specific stains:

     eg   for proteins - Silver stain, or Coomassie brilliant blue
          for nucleic acids - Ethidium bromide (fluorescent)

Types of Supporting Media

Thin film supports

Gels - cast in rectangular slabs or cylinder form

Optimising Electrophoretic Separation

• Faster separation using high voltage because speed of migration proportional to field strength.
  
• But current is proportional to voltage, and excessive current causes heating --> distorts bands by convective mixing and risks denaturing or degrading labile molecules.
  
• Use buffer of low conductivity to minimise current at high voltage.

  Refrigerate buffer for electrophoresis.

  In high voltage electrophoresis, might need additional cooling during run

Agarose Gel Electrophoresis

Agarose
  -   neutral polysaccharide constituent of agar
  -   dissolves in boiling H₂O and forms gel on cooling
  -   very large pore size gel, hence suitable for very large macromolecules especially NUCLEIC ACIDS

Used in RESTRICTION ENZYME MAPPING OF GENE SEQUENCES - to determine sizes of fragments created by cleavage of DNA by restriction endonucleases

Usual set up is horizontal submarine gel

Polyacrylamide Gel Electrophoresis (PAGE)

PAGE is the preferred method for PROTEINS

Gel prepared immediately before use by co-polymerisation of acrylamide and N,N'-methylene bis acrylamide under UV light.

Porosity controlled by proportions of the two components. Larger pore size for larger proteins. Gradient gels also possible.

Gels run horizontally or vertically, either cast as slabs (see below) - with loading wells created by "comb" mold during casting, or as set of cylindrical tubes (one sample/tube)

DISC PAGE

DISCONTINUOUS POLYACRYLAMIDE GEL ELECTROPHORESIS

A means of enhancing the high resolution of PAGE for protein analysis by focussing the sample into very sharp bands before entering the running gel.

Glycine at pH 6.7 has very little net -ve charge, hence low mobility compared to Cl^- and protein^- ions.
Proteins stack into series of very sharp bands in between Cl^- and Gly^- with little separation, then enter running gel

9.7.2  SDS PAGE

SDS = Sodium Dodecyl Sulphate

Isoelectric Focussing

High resolution electrophoretic separation method in which proteins SEPARATE ON BASIS OF DIFFERENCES IN ISOELECTRIC POINT (_PI) by having a STABLE pH GRADIENT in the gel.

Stable pH gradient produced using a MIXTURE of AMPHOLYTES (synthetic polyelectrolytes) in solution. In electric field these migrate until each molecule resides (and exerts buffering action) at a position in gel where the pH is such that it carries zero net charge.

eg   an ampholyte with +VE CHARGE at neutral pH migrates towards CATHODE until its charge is neutralised by OH^- ions moving away from cathode where they form by electrolysis:

2H₂O + 2e^- ----> H₂ + 2OH^-

Sample (mixture of proteins) is subjected to electrophoresis in the pre-formed pH gradient.
Each protein migrates through gel until point where buffered pH equal to its _PI. Protein stops at this position - no further electric force on it.

Bands in gel correspond to protein molecules of different pI

Two-Dimensional Electrophoresis

Protein mixture (eg cell extract) placed at corner of gel slab.

1. Subject to isoelectric focussing - separate proteins by pI differences along one edge of gel.
2. Then turn gel 90^o and subject to SDS PAGE - each pI spot separates on basis of differences in subunit Molecular Mass.

This method produces very characteristic protein maps. Use eg to identify new/abnormal proteins formed by cells in response to metabolic changes.

PROTEIN & NUCLEIC ACID BLOTTING

For sensitive detection of SPECIFIC BANDS after electrophoretic separation.

Separated molecules transferred from gel onto nitrocellulose or similar membrane by BLOTTING.

Detection of bands fixed to membrane.

Southern Blotting: Detect DNA sequences by complementary DNA probe labelled with ³²P or chemiluminescent groups.

Northern Blotting : Detect specific RNA sequences by labelled DNA probe

Western Blotting : Detect specific proteins by monoclonal antibodies