• Charged molecules in an electric field behave in a predictable manner.
  • Positively charged molecules will move towards the negative pole while negatively charged molecules move towards the positive pole = Electrophoresis.
  • Demonstrated by Tiselius (1937) (Nobel Prize in 1948)

Factors which affect the mobility of a substance

      • Electric field strength
      • Sample
      • Buffer and supporting medium

I Electric field strength

  1. Voltage

 rate of migration directly proportional to the potential gradient

in voltage           Potential gradient          mobility

Potential gradient: ratio of distance between the electrode to the potential difference between electrodes


            When potential difference is applied between the electrodes            current is generated.

Current generated     applied voltage

in voltage            Total charge          mobility/unit time

Direct Current from Power pack used


     Flow of current between electrodes depends on

– Supporting medium

– nature of buffer and its concentration

Rate of migration              Resistance

II Sample

  1. Charge of the Sample

Migration               Charge

  1. b) Size

      Smaller the size            Mobility

Larger the size           Mobility (friction &electrostatic forces of                                                     supporting medium and buffer)

  1. c) Shape

            Molecules of llr size but different shape – differ in mobility

Globular proteins migrate faster than fibrous protein                                                      (compact shape)

Super-coiled DNA migrate faster than open circular &linear DNA


III  Buffer

  1. Composition

         Acetate, Borate, Phosphate, Tris-EDTA used

Buffer component must not react/bind with sample

Binding hinders mobility

Binding advantageous for Carbohydrates

(Borate bind to CHO – charged – separated by electrophoresis)

  1. b) Concentration/Ionic strength

            Ionic strength    conc. of  buffer components        Current        carried by   them  (  current carried by sample – slow migration).


High ionic strength buffer —  overall current (    in heat)

            Low ionic strength buffer —   overall current (diffusion of bands – lower resolution)

Optimum conc. : 0.05 – 0.1 M

  1. c) pH

            Determines the extent of ionization (hence the migration rate)

Inorganic sub. (fully ionized) — Less effect

Organic bio-molecules        — Pronounced effect

Continuous buffer system (Supporting medium & Reservoir                                                  buffer same pH) [Agarose]

Discontinuous buffer system (buffers of different pH used) [PAGE, SDS-PAGE]

IV Supporting Medium

            Must be inert, any charge present can affect migration of                               molecules by:


retards mobility of sub. & resolution (tailing)

occurs due to Van der Walls forces, H bonds)


(migration of dissociatable cations of the supporting medium and their associated water molecules towards cathode)

Cathodal movement disrupts normal electrophoresis –               samples move from cathode (-) [Black] to anode (+) [Red] {internal convection}

Types of electrophoresis

Filter paper electrophoresis

Whatman filter Paper/Celluose acetate papers used

    *  Proteins are easily denatured due to the high

absorbance of filter paper

*Works for small peptides or amino acids.

Thin layer electrophoresis (TLE)

– Chemically-modified cellulose/silica/alumina coated slides
Gel electrophoresis

– Starch gel electrophoresis

– Agarose gel electrophoresis (AGE)

– Polyacrylamide Gel Electrophoresis (PAGE)

– Immunoelectrophoresis   (CIE, Laurells Rocket Electrophoresis, 2-D IEP)

Main types of PAGE 


* Enzyme activities are retained after electrophoresis, so          enzymatic assays can be performed on separated proteins

* Factors affecting  mobility:

  • charge
  • molecular weight and the shape of proteins


* SDS (sodium dodecyl sulfate) coats the surface of proteins

* Proteins are denatured, so enzyme activities are lost after   *Factors affecting  mobility :

molecular weight


Visualisation of proteins in gels

Two most commonly used methods are:

  • Coomassie Brilliant staining
  • Silver staining (~1ng of protein per band could be detected)
  • Sypro Ruby stains glycoproteins, lipoproteins and Ca2+ binding proteins and other difficult-to-stain proteins

Polyacrylamide Gels

  • Polyacrilamide is a polymer made of acrylamide (C3H5NO) and bis-acrylamide (N,N’-methylene-bis-acrylamide C7H10N2O2)

Polyacrylamide Gels

  • Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate
  • TEMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction
  • bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains.

What is so special about SDS?

  • SDS is a negatively charged detergent.

(SDS is a common ingredient in detergents, shampoos

laundry detergent and other cleaning products)

Other names for SDS include laurel sulfate and sodium       laurel sulfate

  • Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding protein.
  • ‘Masks’ charge on protein so that all proteins act the same as regards charge.
  • SDS also sticks to proteins in a ratio of approximately 1.4 g of SDS for each gram of protein
  • Prevents protein aggregation.
  • Prevents protein shape from influencing gel run.

Steps in SDS-PAGE

  • Extract Protein
  • Solubilize and Denature Protein
  • Separate Proteins on a gel
  • Stain proteins (visualization)
  • Analyze and interpret results



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