Electrophoresis

Electrophoresis is the migration of charged particle in an electric field towards the electrode bearing the opposite charge.

PRINCIPLEMany important biological molecules such as

aminoacids,peptides,proteins,nucleotides & nucleic acid posses ionizable groups & therefore at any given pH exist in solution as electrically charged species, either as anions (-) or cations (+).Under the influence of an electrical field these charged particles (cations) migrate to the cathode (Negative electrode) or (anions) move to the anode( positive electrode), depending on the nature of their net charge.

FACTORS AFFECTING ELECTROPHORESIS1.Net charge on the molecule Grater charge grater mobility2.Size & shape of molecule Grater size ↓se mobility

Shape Globular -----------↑se mobility

Fibrous ----------- ↓se mobility

3.Ionic strength & pH of buffer

↑ se ionic strength ↑se heat production ↓se mobility

↓se ionic strength less heat production ↑se mobility

pH of buffer 1-11

4.Strength of electric field

5.Temprature

6.Nature of support medium

TYPES OF ELECTROPHORESIS

ELECTROPHORESIS APPARATUS

Electrophoresis tank – Buffer chamber Electrodes Electrical connection to power

supply Space for keeping support

medium Power tank – to provide constant currents or

constant voltage

ELECTROPHORETIC TANK AND POWER PACK

APPARATUS1.Electrophoresis is carried out in a tank suitable for

supporting medium e.g. paper or gel. Major part of tank include – Buffer chamber Electrodes Electrical connection to power supply Space for keeping support medium i.e. paper & slides of agar gel.2.Power pack

SUPPORT MEDIUMFilter paperCellulose acetate membraneAgar & agaroseStarch Polyacrylamide

ELECTROPHORETIC TANK AND POWER PACK

ADDITIONAL REQUIREMENTS

BufferFixativeStaining solutionDestaining solutionDensitometer – is essentially a double beam filter

photometer or spectrophotometer that scans the electrophoretic strip ( in the form of agarose , cellulose acetate or polyacrylamide) as it moves past the optical system.

DENSITOMETER

PROCEDURE1. Sample to be separated is applied to a supporting medium (paper, cellulose acetate, agar gel, polyacrylamide gel etc.)

2. Electrophoresis is carried out at desired constant voltage or constant current in presence of specific pH.

3. After completion of electrophoresis the supporting medium is placed in a fixative to prevent diffusion of separated fractions.

4. Separated fraction is then visualized by using appropriate stains e.g. Bromophenol Blue & Amino Schwartz for plasma proteins & Sudan Black for lipoproteins.

5. Quantification of each fraction is done by either densitometer or elution followed by colorimeter or spectrophotometer of eluted fraction.

RUNNING OF ELECTROPHORESIS

APPLICATIONS

1.Separation of biological molecules like plasma proteins , nucleic acids, nucleotides, charged carbohydrate derivatives,glycoproteins,lipoproteins, hemoglobin variants, isoenzymes etc.

2.Analytical Applications-Determination of DNA sequencesSouthern & Northern BlottingRestriction Mapping of DNA3. In Protein StudyDetermination of subunit stoichiometryDetermination of molecular weight

TECHNIQUEAPPICATION OF SAMPLERUNNING OF SAMPLEVISUALISATION OF SAMPLEQUANTIFICATION

APPLICATIONSTypes of electrophoresis

Support medium applications

paper Whatman’s paper no.1 Separation of peptidesPlasma proteinsNucleic acidCharged carbohydrates

Cellulose acetate Cellulose acetate strips in which hydroxyl gr.of cellulose is acetate with acetic anhydride

Separation of GlycoproteinsLipoproteinsHb derivatives

Gel :- Based on charge & molecular size

a. Agarose gel Composed of agarose & agaropectin

Separation of plasma proteinsLipoproteinsHb derivatives

CONTINUED b. Starch Partially hydrolysed

starchSeparation of isoenzymes

c. polyacrylamide Polyacrylamide gel layers of varying pore size

Separation of GlycoproteinsLipoproteinsHb derivativesDNA fragments

Isoelectricfocussing Polyacrylamide gel Separation of plasma proteinsLipoproteinsHb derivatives

Immunoelectrophoresis Agar gel slab Separation of antigenic proteins

TYPES OF PLASMA PROTEINSAlbumin Globulins- α1, α2, β,γ globulins

How To write in journalDefinationFactors affecting electrophoresisTypes of electrophoresisProcedureApplications